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Petrova IO, Smirnikhina SA. Prime Editing in Dividing and Quiescent Cells. Int J Mol Sci 2025; 26:3596. [PMID: 40332080 PMCID: PMC12026808 DOI: 10.3390/ijms26083596] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2025] [Revised: 03/29/2025] [Accepted: 04/01/2025] [Indexed: 05/08/2025] Open
Abstract
Prime editing is a method of genome editing based on reverse transcription. Recent results have shown its elevated efficiency in dividing cells, which raises some questions regarding the mechanism of this effect, because prime editing does not employ homology-driven repair. This mini review aims to identify the reason for this phenomenon and find a possible solution to the problems that it poses. In dividing cells, prime editing takes advantage of high levels of dNTPs and active endonuclease and ligase machinery, such as FEN1 endonuclease and LIG1 ligase, but DNA mismatch repair, which is closely associated with replication, works against prime editing. Prime editing is a method which relies on retroviral reverse transcription, so mechanisms of intrinsic anti-retroviral defense should also work against editing. One of the factors which drastically reduce the efficiency of reverse translation is SAMHD1, which maintains low levels of dNTPs in non-dividing cells. Recent works aimed at the mitigation of SAMHD1 function demonstrated a significant increase in prime editing efficiency.
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Affiliation(s)
- Irina O. Petrova
- Laboratory of Genome Editing, Research Center for Medical Genetics, Moskvorechye 1, 115478 Moscow, Russia
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2
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Wang N, Zhang S, Langfelder P, Ramanathan L, Gao F, Plascencia M, Vaca R, Gu X, Deng L, Dionisio LE, Vu H, Maciejewski E, Ernst J, Prasad BC, Vogt TF, Horvath S, Aaronson JS, Rosinski J, Yang XW. Distinct mismatch-repair complex genes set neuronal CAG-repeat expansion rate to drive selective pathogenesis in HD mice. Cell 2025; 188:1524-1544.e22. [PMID: 39938516 PMCID: PMC11972609 DOI: 10.1016/j.cell.2025.01.031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Revised: 01/09/2025] [Accepted: 01/21/2025] [Indexed: 02/14/2025]
Abstract
Huntington's disease (HD) modifiers include mismatch-repair (MMR) genes, but their connections to neuronal pathogenesis remain unclear. Here, we genetically tested 9 HD genome-wide association study (GWAS)/MMR genes in mutant Huntingtin (mHtt) mice with 140 inherited CAG repeats (Q140). Knockout (KO) of genes encoding a distinct MMR complex either strongly (Msh3 and Pms1) or moderately (Msh2 and Mlh1) rescues phenotypes with early onset in striatal medium-spiny neurons (MSNs) and late onset in the cortical neurons: somatic CAG-repeat expansion, transcriptionopathy, and mHtt aggregation. Msh3 deficiency ameliorates open-chromatin dysregulation in Q140 neurons. Mechanistically, the fast linear rate of mHtt modal-CAG-repeat expansion in MSNs (8.8 repeats/month) is drastically reduced or stopped by MMR mutants. Msh3 or Pms1 deficiency prevents mHtt aggregation by keeping somatic MSN CAG length below 150. Importantly, Msh3 deficiency corrects synaptic, astrocytic, and locomotor defects in HD mice. Thus, Msh3 and Pms1 drive fast somatic mHtt CAG-expansion rates in HD-vulnerable neurons to elicit repeat-length/threshold-dependent, selective, and progressive pathogenesis in vivo.
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Affiliation(s)
- Nan Wang
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Shasha Zhang
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Peter Langfelder
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Lalini Ramanathan
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Fuying Gao
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Mary Plascencia
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Raymond Vaca
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Xiaofeng Gu
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Linna Deng
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Leonardo E Dionisio
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
| | - Ha Vu
- Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA
| | - Emily Maciejewski
- Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA
| | - Jason Ernst
- Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, CA, USA
| | | | | | - Steve Horvath
- Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA; Altos Labs, Cambridge, UK
| | | | | | - X William Yang
- Center for Neurobehavioral Genetics, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA; Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
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3
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Fukui K, Murakawa T, Hino N, Kondo N, Yano T. ATP binding controls the molecular function of bacterial MutS2 by mediating closure of the dimeric clamp structure. Structure 2025:S0969-2126(25)00100-5. [PMID: 40157362 DOI: 10.1016/j.str.2025.03.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Revised: 02/16/2025] [Accepted: 03/04/2025] [Indexed: 04/01/2025]
Abstract
MutS2 recognizes branched DNA structures to regulate homologous recombination. MutS2 also has a role in ribosome recycling, where it resolves collided ribosomes. These functions require ATP-dependent conformational changes of MutS2. In the known nucleotide-free and ADP-bound MutS2 structures the dimeric clamp-like structure adopts open conformations. Here, we present the crystal structure of MutS2 with a bound ATP analog revealing a closed conformation of the clamp. Experiments with MutS2, where an unnatural photo-crosslinking capable amino acid was introduced into the clamp revealed that ATP-dependent closure also occurs in solution. Binding of MutS2 to a terminal-containing DNA was not affected by ATP, whereas that to a terminal-less DNA was reinforced. These findings suggest that clamp closure enables MutS2 to stay bound to recombination intermediates, which might regulate recombination. Furthermore, closure of the clamp provides insights into the mechanism of dissociation of collided ribosomes mediated by MutS2.
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Affiliation(s)
- Kenji Fukui
- Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.
| | - Takeshi Murakawa
- Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan
| | - Nobumasa Hino
- Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; Center for Infectious Disease Education and Research, Osaka University, 2-8 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Naoyuki Kondo
- Department of Molecular Genetics, Institute of Biomedical Science, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka 573-1010, Japan
| | - Takato Yano
- Department of Biochemistry, Faculty of Medicine, Osaka Medical and Pharmaceutical University, 2-7 Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.
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4
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Casazza KM, Williams GM, Johengen L, Twoey G, Surtees JA. Msh2-Msh3 DNA-binding is not sufficient to promote trinucleotide repeat expansions in Saccharomyces cerevisiae. Genetics 2025; 229:iyae222. [PMID: 39790027 PMCID: PMC11912836 DOI: 10.1093/genetics/iyae222] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2024] [Accepted: 12/25/2024] [Indexed: 01/12/2025] Open
Abstract
Mismatch repair (MMR) is a highly conserved DNA repair pathway that recognizes mispairs that occur spontaneously during DNA replication and coordinates their repair. In Saccharomyces cerevisiae, Msh2-Msh3 and Msh2-Msh6 initiate MMR by recognizing and binding insertion or deletion (in/del) loops up to ∼17 nucleotides (nt.) and base-base mispairs, respectively; the 2 complexes have overlapping specificity for small (1-2 nt.) in/dels. The DNA-binding specificity for the 2 complexes resides in their respective mispair binding domains (MBDs) and has distinct DNA-binding modes. Msh2-Msh3 also plays a role in promoting CAG/CTG trinucleotide repeat (TNR) expansions, which underlie many neurodegenerative diseases such as Huntington's disease and myotonic dystrophy type 1. Models for Msh2-Msh3's role in promoting TNR tract expansion have invoked its specific DNA-binding activity and predict that the TNR structure alters its DNA binding and downstream activities to block repair. Using a chimeric Msh complex that replaces the MBD of Msh6 with the Msh3 MBD, we demonstrate that Msh2-Msh3 DNA-binding activity is not sufficient to promote TNR expansions. We propose a model for Msh2-Msh3-mediated TNR expansions that requires a fully functional Msh2-Msh3 including DNA binding, coordinated ATP binding, and hydrolysis activities and interactions with Mlh complexes that are analogous to those required for MMR.
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Affiliation(s)
- Katherine M Casazza
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Gregory M Williams
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
- Curia Global, Inc., Buffalo, NY 14203, USA
| | - Lauren Johengen
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Gavin Twoey
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
| | - Jennifer A Surtees
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14203, USA
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Jimenez DA, Walker A, Usdin K, Zhao X. Tissue-Specific Effects of the DNA Helicase FANCJ/BRIP1/BACH1 on Repeat Expansion in a Mouse Model of the Fragile X-Related Disorders. Int J Mol Sci 2025; 26:2655. [PMID: 40141297 PMCID: PMC11942155 DOI: 10.3390/ijms26062655] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2025] [Revised: 03/06/2025] [Accepted: 03/07/2025] [Indexed: 03/28/2025] Open
Abstract
Fragile X-related disorders (FXDs) are caused by the expansion of a CGG repeat tract in the 5'-UTR of the FMR1 gene. The expansion mechanism is likely shared with the 45+ other human diseases resulting from repeat expansion, a process that has been shown to require key mismatch repair (MMR) factors. FANCJ, a DNA helicase involved in unwinding unusual DNA secondary structures, has been implicated in a number of DNA repair processes including MMR. To test the role of FANCJ in repeat expansion, we crossed FancJ-null mice to an FXD mouse model. We found that loss of FANCJ resulted in a trend towards more extensive expansion that was significant for the small intestine and male germline. This finding has interesting implications for the expansion mechanism and raises the possibility that other DNA helicases may be important modifiers of expansion risk in certain cell types.
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Affiliation(s)
| | | | - Karen Usdin
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; (D.A.J.); (A.W.)
| | - Xiaonan Zhao
- Section on Gene Structure and Disease, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; (D.A.J.); (A.W.)
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Duan J, Chen T, Li Q, Zhang Y, Lu T, Xue J, Sun Y, Gao L, Zhang Y. Protein arginine methyltransferase 6 enhances immune checkpoint blockade efficacy via the STING pathway in MMR-proficient colorectal cancer. J Immunother Cancer 2025; 13:e010639. [PMID: 40086819 PMCID: PMC11907083 DOI: 10.1136/jitc-2024-010639] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2024] [Accepted: 02/26/2025] [Indexed: 03/16/2025] Open
Abstract
BACKGROUND The emergence of immunotherapy has revolutionized the paradigm of cancer treatment with immune checkpoint blockades (ICB) in solid cancers, including colorectal cancer (CRC). However, only a small subset of CRC patients harboring deficient mismatch repair (dMMR) or microsatellite instability-high (MSI-H) benefits from ICB therapy. A very limited response to ICB therapy has been achieved in MMR-proficient CRC, representing a significant challenge limiting the clinical application of immunotherapy. MMR is the critical DNA repair pathway that maintains genomic integrity by correcting DNA mismatches, which is mediated by the MutSα or MutSβ complex consisting of MSH2 with MSH6 and MSH3, respectively. Given that MMR status directs effective immune response, we sought to determine whether targeting MMR capacity boosts ICB efficacy. METHODS Azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC and xenograft model were used to evaluate the function of PRMT6 and response to PRMT6 inhibitor EPZ020411 and combination therapy of PD1 and EPZ020411. Biochemical assays were performed to elucidate the underlying mechanism of PRMT6-mediated MSH2 methylation and immune evasion. RESULTS We have identified PRMT6 as a crucial regulator of MMR capacity via MSH2 dimethylation at R171 and R219. Such a modification abrogates its MMR capacity and prevents the recruitment of MSH3 and MSH6. PRMT6 loss or inhibition triggers cytosolic DNA accumulation and cGAS-STING signaling activation, leading to enhanced immune response in PRMT6-deficient colon tumors or xenografts. Pharmacological inhibition of PRMT6 using EPZ020411 promotes mutagenesis and destabilizes MutSα or MutSβ assembly, and prolonged EPZ020411 exposure maintains an MSI-like phenotype in microsatellite stability (MSS) cells. EPZ020411 treatment sensitizes ICB efficacy of MSS cells, but not MSI cells in vivo. Similar effects have been observed in MSS colon tumors induced by AOM/DSS. CONCLUSIONS Our study provides a preclinical proof of concept to overcome resistance to immunotherapy by targeting PRMT6 in CRC with MSS.
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Affiliation(s)
- Jinlin Duan
- Department of General Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
- Department of Pathology, Tongren Hospital Shanghai Jiaotong University School of Medicine, Shanghai, China
| | - Tao Chen
- Department of Biliary-Pancreatic Surgery, Shanghai Jiao Tong University, Shanghai, China
| | - Qiwei Li
- Department of Biliary-Pancreatic Surgery, Shanghai Jiao Tong University, Shanghai, China
| | - Yu Zhang
- Department of Clinical Laboratory, Shanghai Sixth People's Hospital, Shanghai, China
- Department of Clinical Laboratory, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
| | - Ting Lu
- Department of Clinical Laboratory, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
| | - Junyan Xue
- Department of Clinical Laboratory, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
| | - Yang Sun
- Department of Clinical Laboratory, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
| | - Ling Gao
- Department of General Surgery, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, China
| | - Yonglong Zhang
- Laboratory of Targeted Therapy and Precision Medicine, Department of Clinical Laboratory, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
- Department of General Surgery, Shanghai 6th Peoples Hospital Affiliated to Shanghai Jiao Tong University, Shanghai, China
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7
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Marzec P, Richer M, Lahue RS. Therapeutic targeting of mismatch repair proteins in triplet repeat expansion diseases. DNA Repair (Amst) 2025; 147:103817. [PMID: 40010080 DOI: 10.1016/j.dnarep.2025.103817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2024] [Revised: 02/07/2025] [Accepted: 02/07/2025] [Indexed: 02/28/2025]
Abstract
Triplet repeat expansion diseases are a class of ∼20 inherited neurological disorders. Many of these diseases are debilitating, sometimes fatally so, and they have unfortunately proved difficult to treat. New compelling evidence shows that somatic repeat expansions in some diseases are essential to the pathogenic process, accelerating the age of onset and the rate of disease progression. Inhibiting somatic repeat expansions, therefore, provides a therapeutic opportunity to delay or block disease onset and/or slow progression. Several key aspects enhance the appeal of this therapeutic approach. First, the proteins responsible for promoting expansions are known from human genetics and model systems, obviating the need for lengthy target searches. They include the mismatch repair proteins MSH3, PMS1 and MLH3. Second, inhibiting or downregulating any of these three proteins is attractive due to their good safety profiles. Third, having three potential targets helps mitigate risk. Fourth, another protein, the nuclease FAN1, protects against expansions; in principle, increasing FAN1 activity could be therapeutic. Fifth, therapies aimed at inhibiting somatic repeat expansions could be used against several diseases that display this shared mechanistic feature, offering the opportunity for one treatment against multiple diseases. This review will address the underlying findings and the recent therapeutic advances in targeting MSH3, PMS1, MLH3 and FAN1 in triplet repeat expansion diseases.
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Affiliation(s)
- Paulina Marzec
- LoQus23 Therapeutics Ltd, Cambridge CB22 3AT, United Kingdom
| | | | - Robert S Lahue
- Centre for Chromosome Biology, University of Galway, H91W2TY, Ireland; Galway Neuroscience Centre, University of Galway, H91W2TY, Ireland.
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Ma X, Li F, Chen Q, Gao S, Bai F. NesT-NABind: a Nested Transformer for Nucleic Acid-Binding Site Prediction on Protein Surface. J Chem Inf Model 2025; 65:1166-1177. [PMID: 39818834 DOI: 10.1021/acs.jcim.4c01765] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2025]
Abstract
Protein-nucleic acid interactions play a crucial role in many physiological processes. Identifying the binding sites of nucleotides on the protein surface is the prerequisite for understanding the molecular recognition mechanisms between the two types of macromolecules and also provides the information to design or generate molecule modulators against these sites to manipulate biological function according to specific requirements. Existing studies mainly focus on characterizing local surfaces around sites, often neglecting the interrelationships among these sites and the global protein information. To address this gap, we propose NesT-NABind, a Nested Transformer for Nucleic Acid-Binding site prediction. This model leverages the Transformer's advanced capabilities in contextual understanding and long-range dependency capturing. Specifically, we introduce a local patch-scale Transformer to process surface information around each site and a global protein-scale transformer to integrate surface and sequence information on the entire protein. These two Transformers operate at different scales of protein, hence the term "nested". Experiments demonstrate that NesT-NABind achieves a 5.57% improvement in the F1 score and a 3.64% improvement in AUPRC compared to state-of-the-art methods. With the incorporation of global features, NesT-NABind shows an enhanced predictive capability for the challenging large proteins and therefore can be used in a much wider range of applications.
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Affiliation(s)
- Xinyue Ma
- School of Information Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
| | - Fenglei Li
- School of Information Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- Department of Computer Science, Aalto University,Konemiehentie 2, Espoo02150,Finland
| | - Qianyu Chen
- School of Information Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
| | - Shenghua Gao
- Department of Computer Science, The University of Hong Kong, Pokfolam Road, HKSAR, 999077, China
- HKU Shanghai lntelligent Computing Research Center, Shanghai, 201210, China
| | - Fang Bai
- School of Information Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New Area, Shanghai 201210, China
- School of Life Science and Technology, ShanghaiTech University, Pudong New Area, 393 Middle Huaxia Road, Shanghai 201210, China
- Shanghai Clinical Research and Trial Center, No.1599 Keyuan Road, Pudong New Area, Shanghai 201210, China
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Hujoel MLA, Handsaker RE, Kamitaki N, Mukamel RE, Rubinacci S, Palamara PF, McCarroll SA, Loh PR. Insights into the causes and consequences of DNA repeat expansions from 700,000 biobank participants. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.25.625248. [PMID: 39651202 PMCID: PMC11623664 DOI: 10.1101/2024.11.25.625248] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2024]
Abstract
Expansions and contractions of tandem DNA repeats are a source of genetic variation in human populations and in human tissues: some expanded repeats cause inherited disorders, and some are also somatically unstable. We analyzed DNA sequence data, derived from the blood cells of >700,000 participants in UK Biobank and the All of Us Research Program, and developed new computational approaches to recognize, measure and learn from DNA-repeat instability at 15 highly polymorphic CAG-repeat loci. We found that expansion and contraction rates varied widely across these 15 loci, even for alleles of the same length; repeats at different loci also exhibited widely variable relative propensities to mutate in the germline versus the blood. The high somatic instability of TCF4 repeats enabled a genome-wide association analysis that identified seven loci at which inherited variants modulate TCF4 repeat instability in blood cells. Three of the implicated loci contained genes ( MSH3 , FAN1 , and PMS2 ) that also modulate Huntington's disease age-at-onset as well as somatic instability of the HTT repeat in blood; however, the specific genetic variants and their effects (instability-increasing or-decreasing) appeared to be tissue-specific and repeat-specific, suggesting that somatic mutation in different tissues-or of different repeats in the same tissue-proceeds independently and under the control of substantially different genetic variation. Additional modifier loci included DNA damage response genes ATAD5 and GADD45A . Analyzing DNA repeat expansions together with clinical data showed that inherited repeats in the 5' UTR of the glutaminase ( GLS) gene are associated with stage 5 chronic kidney disease (OR=14.0 [5.7-34.3]) and liver diseases (OR=3.0 [1.5-5.9]). These and other results point to the dynamics of DNA repeats in human populations and across the human lifespan.
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10
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Pan F, Xu P, Roland C, Sagui C, Weninger K. Structural and Dynamical Properties of Nucleic Acid Hairpins Implicated in Trinucleotide Repeat Expansion Diseases. Biomolecules 2024; 14:1278. [PMID: 39456210 PMCID: PMC11505666 DOI: 10.3390/biom14101278] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2024] [Revised: 09/26/2024] [Accepted: 10/05/2024] [Indexed: 10/28/2024] Open
Abstract
Dynamic mutations in some human genes containing trinucleotide repeats are associated with severe neurodegenerative and neuromuscular disorders-known as Trinucleotide (or Triplet) Repeat Expansion Diseases (TREDs)-which arise when the repeat number of triplets expands beyond a critical threshold. While the mechanisms causing the DNA triplet expansion are complex and remain largely unknown, it is now recognized that the expandable repeats lead to the formation of nucleotide configurations with atypical structural characteristics that play a crucial role in TREDs. These nonstandard nucleic acid forms include single-stranded hairpins, Z-DNA, triplex structures, G-quartets and slipped-stranded duplexes. Of these, hairpin structures are the most prolific and are associated with the largest number of TREDs and have therefore been the focus of recent single-molecule FRET experiments and molecular dynamics investigations. Here, we review the structural and dynamical properties of nucleic acid hairpins that have emerged from these studies and the implications for repeat expansion mechanisms. The focus will be on CAG, GAC, CTG and GTC hairpins and their stems, their atomistic structures, their stability, and the important role played by structural interrupts.
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Affiliation(s)
- Feng Pan
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
- Department of Statistics, Florida State University, Tallahassee, FL 32306, USA
| | - Pengning Xu
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Christopher Roland
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
| | - Celeste Sagui
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
| | - Keith Weninger
- Department of Physics, North Carolina State University, Raleigh, NC 27695, USA; (F.P.); (C.R.)
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11
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Wang N, Zhang S, Langfelder P, Ramanathan L, Plascencia M, Gao F, Vaca R, Gu X, Deng L, Dionisio LE, Prasad BC, Vogt T, Horvath S, Aaronson JS, Rosinski J, Yang XW. Msh3 and Pms1 Set Neuronal CAG-repeat Migration Rate to Drive Selective Striatal and Cortical Pathogenesis in HD Mice. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.09.602815. [PMID: 39026894 PMCID: PMC11257559 DOI: 10.1101/2024.07.09.602815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/20/2024]
Abstract
Modifiers of Huntington's disease (HD) include mismatch repair (MMR) genes; however, their underlying disease-altering mechanisms remain unresolved. Knockout (KO) alleles for 9 HD GWAS modifiers/MMR genes were crossed to the Q140 Huntingtin (mHtt) knock-in mice to probe such mechanisms. Four KO mice strongly ( Msh3 and Pms1 ) or moderately ( Msh2 and Mlh1 ) rescue a triad of adult-onset, striatal medium-spiny-neuron (MSN)-selective phenotypes: somatic Htt DNA CAG-repeat expansion, transcriptionopathy, and mHtt protein aggregation. Comparatively, Q140 cortex also exhibits an analogous, but later-onset, pathogenic triad that is Msh3 -dependent. Remarkably, Q140/homozygous Msh3-KO lacks visible mHtt aggregates in the brain, even at advanced ages (20-months). Moreover, Msh3 -deficiency prevents striatal synaptic marker loss, astrogliosis, and locomotor impairment in HD mice. Purified Q140 MSN nuclei exhibit highly linear age-dependent mHtt DNA repeat expansion (i.e. repeat migration), with modal-CAG increasing at +8.8 repeats/month (R 2 =0.98). This linear rate is reduced to 2.3 and 0.3 repeats/month in Q140 with Msh3 heterozygous and homozygous alleles, respectively. Our study defines somatic Htt CAG-repeat thresholds below which there are no detectable mHtt nuclear or neuropil aggregates. Mild transcriptionopathy can still occur in Q140 mice with stabilized Htt 140-CAG repeats, but the majority of transcriptomic changes are due to somatic repeat expansion. Our analysis reveals 479 genes with expression levels highly correlated with modal-CAG length in MSNs. Thus, our study mechanistically connects HD GWAS genes to selective neuronal vulnerability in HD, in which Msh3 and Pms1 set the linear rate of neuronal mHtt CAG-repeat migration to drive repeat-length dependent pathogenesis; and provides a preclinical platform for targeting these genes for HD suppression across brain regions. One Sentence Summary Msh3 and Pms1 are genetic drivers of sequential striatal and cortical pathogenesis in Q140 mice by mediating selective CAG-repeat migration in HD vulnerable neurons.
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12
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Peñafiel-Ayala A, Peralta-Castro A, Mora-Garduño J, García-Medel P, Zambrano-Pereira AG, Díaz-Quezada C, Abraham-Juárez MJ, Benítez-Cardoza CG, Sloan DB, Brieba LG. Plant Organellar MSH1 Is a Displacement Loop-Specific Endonuclease. PLANT & CELL PHYSIOLOGY 2024; 65:560-575. [PMID: 37756637 PMCID: PMC11494383 DOI: 10.1093/pcp/pcad112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/16/2023] [Revised: 09/09/2023] [Accepted: 09/19/2023] [Indexed: 09/29/2023]
Abstract
MutS HOMOLOG 1 (MSH1) is an organellar-targeted protein that obstructs ectopic recombination and the accumulation of mutations in plant organellar genomes. MSH1 also modulates the epigenetic status of nuclear DNA, and its absence induces a variety of phenotypic responses. MSH1 is a member of the MutS family of DNA mismatch repair proteins but harbors an additional GIY-YIG nuclease domain that distinguishes it from the rest of this family. How MSH1 hampers recombination and promotes fidelity in organellar DNA inheritance is unknown. Here, we elucidate its enzymatic activities by recombinantly expressing and purifying full-length MSH1 from Arabidopsis thaliana (AtMSH1). AtMSH1 is a metalloenzyme that shows a strong binding affinity for displacement loops (D-loops). The DNA-binding abilities of AtMSH1 reside in its MutS domain and not in its GIY-YIG domain, which is the ancillary nickase of AtMSH1. In the presence of divalent metal ions, AtMSH1 selectively executes multiple incisions at D-loops, but not other DNA structures including Holliday junctions or dsDNA, regardless of the presence or absence of mismatches. The selectivity of AtMSH1 to dismantle D-loops supports the role of this enzyme in preventing recombination between short repeats. Our results suggest that plant organelles have evolved novel DNA repair routes centered around the anti-recombinogenic activity of MSH1.
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Affiliation(s)
- Alejandro Peñafiel-Ayala
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Antolin Peralta-Castro
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Josue Mora-Garduño
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Paola García-Medel
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Angie G Zambrano-Pereira
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Corina Díaz-Quezada
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - María Jazmín Abraham-Juárez
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
| | - Claudia G Benítez-Cardoza
- Laboratorio de Investigación Bioquímica, Programa Institucional en Biomedicina Molecular ENMyH-IPN, Guillermo Massieu Helguera No. 239, La Escalera Ticoman 07320 DF, México
| | - Daniel B Sloan
- Department of Biology, Colorado State University, Fort Collins, CO 80523, USA
| | - Luis G Brieba
- Langebio-Cinvestav Sede Irapuato, Km. 9.6 Libramiento Norte Carretera. Irapuato-León, Irapuato, Guanajuato 36821, México
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13
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Isik E, Shukla K, Pospisilova M, König C, Andrs M, Rao S, Rosano V, Dobrovolna J, Krejci L, Janscak P. MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops. SCIENCE ADVANCES 2024; 10:eadk2685. [PMID: 38324687 PMCID: PMC10849593 DOI: 10.1126/sciadv.adk2685] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/11/2023] [Accepted: 01/08/2024] [Indexed: 02/09/2024]
Abstract
Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSβ, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSβ, MutLβ, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.
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Affiliation(s)
- Esin Isik
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Kaustubh Shukla
- Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 143 00 Prague, Czech Republic
| | - Michaela Pospisilova
- Department of Biology and National Centre for Biomolecular Research, Masaryk University, Kamenice 5/A7, Brno 62500, Czech Republic
- International Clinical Research Center, St Anne's University Hospital, Pekarska 53, Brno 656 91, Czech Republic
| | - Christiane König
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Martin Andrs
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Satyajeet Rao
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Vinicio Rosano
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
| | - Jana Dobrovolna
- Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 143 00 Prague, Czech Republic
| | - Lumir Krejci
- Department of Biology and National Centre for Biomolecular Research, Masaryk University, Kamenice 5/A7, Brno 62500, Czech Republic
- International Clinical Research Center, St Anne's University Hospital, Pekarska 53, Brno 656 91, Czech Republic
| | - Pavel Janscak
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
- Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 143 00 Prague, Czech Republic
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14
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Waters KL, Spratt DE. New Discoveries on Protein Recruitment and Regulation during the Early Stages of the DNA Damage Response Pathways. Int J Mol Sci 2024; 25:1676. [PMID: 38338953 PMCID: PMC10855619 DOI: 10.3390/ijms25031676] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 01/26/2024] [Accepted: 01/28/2024] [Indexed: 02/12/2024] Open
Abstract
Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated.
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Affiliation(s)
| | - Donald E. Spratt
- Gustaf H. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610, USA;
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15
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Koi M, Leach BH, McGee S, Tseng-Rogenski SS, Burke CA, Carethers JM. Compound heterozygous MSH3 germline variants and associated tumor somatic DNA mismatch repair dysfunction. NPJ Precis Oncol 2024; 8:12. [PMID: 38243056 PMCID: PMC10798947 DOI: 10.1038/s41698-024-00511-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2023] [Accepted: 12/08/2023] [Indexed: 01/21/2024] Open
Abstract
We describe here an individual from a fourth family with germline compound heterozygous MSH3 germline variants and its observed biological consequences. The patient was initially diagnosed with invasive moderately-differentiated adenocarcinoma of the colon at the age of 43. Germline multigene panel testing revealed a pathogenic variant MSH3 c.2436-1 G > A and a variant of (initial) uncertain significance MSH3 c.3265 A > T (p.Lys1089*). Germline genetic testing of family members confirm the variants are in trans with the c.2436-1 G > A variant of paternal and the c.3265 A > T variant of maternal origin. Tumor DNA exhibits low levels of microsatellite instability and elevated microsatellite alterations at selected tetranucleotide repeats (EMAST). Tissue immunohistochemical staining for MSH3 demonstrated variant MSH3 protein is present in the cytoplasm and cell membrane but not in the nucleus of normal and tumor epithelial cells. Furthermore, variant MSH3 is accompanied by loss of nuclear MSH6 and a reduced level of nuclear MSH2 in some tumor cells, suggesting that the variant MSH3 protein may inhibit binding of MSH6 to MSH2.
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Affiliation(s)
- Minoru Koi
- Division of Gastroenterology & Hepatology, Department of Internal Medicine, and Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
- Division of Gastroenterology & Hepatology, Department of Medicine, and Moores Cancer Center, University of California at San Diego, San Diego, CA, USA
| | - Brandie H Leach
- Center for Personalized Genetic Healthcare, Genomic Medicine Institute, Cleveland Clinic, Cleveland, OH, USA
- Sanford R. Weiss MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Cleveland, OH, USA
| | - Sarah McGee
- Center for Personalized Genetic Healthcare, Genomic Medicine Institute, Cleveland Clinic, Cleveland, OH, USA
- Sanford R. Weiss MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Cleveland, OH, USA
| | - Stephanie S Tseng-Rogenski
- Division of Gastroenterology & Hepatology, Department of Internal Medicine, and Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA
| | - Carol A Burke
- Sanford R. Weiss MD Center for Hereditary Colorectal Neoplasia, Cleveland Clinic, Cleveland, OH, USA
- Department of Gastroenterology, Hepatology and Nutrition, Cleveland Clinic, Cleveland, OH, USA
| | - John M Carethers
- Division of Gastroenterology & Hepatology, Department of Internal Medicine, and Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA.
- Division of Gastroenterology & Hepatology, Department of Medicine, and Moores Cancer Center, University of California at San Diego, San Diego, CA, USA.
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16
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Vu TV, Nguyen NT, Kim J, Hong JC, Kim J. Prime editing: Mechanism insight and recent applications in plants. PLANT BIOTECHNOLOGY JOURNAL 2024; 22:19-36. [PMID: 37794706 PMCID: PMC10754014 DOI: 10.1111/pbi.14188] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 09/14/2023] [Accepted: 09/18/2023] [Indexed: 10/06/2023]
Abstract
Prime editing (PE) technology utilizes an extended prime editing guide RNA (pegRNA) to direct a fusion peptide consisting of nCas9 (H840) and reverse transcriptase (RT) to a specific location in the genome. This enables the installation of base changes at the targeted site using the extended portion of the pegRNA through RT activity. The resulting product of the RT reaction forms a 3' flap, which can be incorporated into the genomic site through a series of biochemical steps involving DNA repair and synthesis pathways. PE has demonstrated its effectiveness in achieving almost all forms of precise gene editing, such as base conversions (all types), DNA sequence insertions and deletions, chromosomal translocation and inversion and long DNA sequence insertion at safe harbour sites within the genome. In plant science, PE could serve as a groundbreaking tool for precise gene editing, allowing the creation of desired alleles to improve crop varieties. Nevertheless, its application has encountered limitations due to efficiency constraints, particularly in dicotyledonous plants. In this review, we discuss the step-by-step mechanism of PE, shedding light on the critical aspects of each step while suggesting possible solutions to enhance its efficiency. Additionally, we present an overview of recent advancements and future perspectives in PE research specifically focused on plants, examining the key technical considerations of its applications.
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Affiliation(s)
- Tien V. Vu
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinjuKorea
| | - Ngan Thi Nguyen
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinjuKorea
| | - Jihae Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinjuKorea
| | - Jong Chan Hong
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinjuKorea
| | - Jae‐Yean Kim
- Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research CenterGyeongsang National UniversityJinjuKorea
- Division of Life ScienceGyeongsang National UniversityJinjuKorea
- Nulla Bio Inc.JinjuKorea
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17
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Rajagopal S, Donaldson J, Flower M, Hensman Moss DJ, Tabrizi SJ. Genetic modifiers of repeat expansion disorders. Emerg Top Life Sci 2023; 7:325-337. [PMID: 37861103 PMCID: PMC10754329 DOI: 10.1042/etls20230015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Revised: 09/20/2023] [Accepted: 10/09/2023] [Indexed: 10/21/2023]
Abstract
Repeat expansion disorders (REDs) are monogenic diseases caused by a sequence of repetitive DNA expanding above a pathogenic threshold. A common feature of the REDs is a strong genotype-phenotype correlation in which a major determinant of age at onset (AAO) and disease progression is the length of the inherited repeat tract. Over a disease-gene carrier's life, the length of the repeat can expand in somatic cells, through the process of somatic expansion which is hypothesised to drive disease progression. Despite being monogenic, individual REDs are phenotypically variable, and exploring what genetic modifying factors drive this phenotypic variability has illuminated key pathogenic mechanisms that are common to this group of diseases. Disease phenotypes are affected by the cognate gene in which the expansion is found, the location of the repeat sequence in coding or non-coding regions and by the presence of repeat sequence interruptions. Human genetic data, mouse models and in vitro models have implicated the disease-modifying effect of DNA repair pathways via the mechanisms of somatic mutation of the repeat tract. As such, developing an understanding of these pathways in the context of expanded repeats could lead to future disease-modifying therapies for REDs.
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Affiliation(s)
- Sangeerthana Rajagopal
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, U.K
- UK Dementia Research Institute, University College London, London WCC1N 3BG, U.K
| | - Jasmine Donaldson
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, U.K
- UK Dementia Research Institute, University College London, London WCC1N 3BG, U.K
| | - Michael Flower
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, U.K
- UK Dementia Research Institute, University College London, London WCC1N 3BG, U.K
| | - Davina J Hensman Moss
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, U.K
- UK Dementia Research Institute, University College London, London WCC1N 3BG, U.K
- St George's University of London, London SW17 0RE, U.K
| | - Sarah J Tabrizi
- UCL Huntington's Disease Centre, Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, Queen Square, London WC1N 3BG, U.K
- UK Dementia Research Institute, University College London, London WCC1N 3BG, U.K
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18
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Li J, Wang H, Yang W. Tandem MutSβ binding to long extruded DNA trinucleotide repeats underpins pathogenic expansions. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.12.571350. [PMID: 38168405 PMCID: PMC10760016 DOI: 10.1101/2023.12.12.571350] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2024]
Abstract
Expansion of trinucleotide repeats causes Huntington's disease, Fragile X syndrome and over twenty other monogenic disorders1. How mismatch repair protein MutSβ and large repeats of CNG (N=A, T, C or G) cooperate to drive the expansion is poorly understood. Contrary to expectations, we find that MutSβ prefers to bind the stem of an extruded (CNG) hairpin rather than the hairpin end or hairpin-duplex junction. Structural analyses reveal that in the presence of MutSβ, CNG repeats with N:N mismatches adopt a B form-like pseudo-duplex, with one or two CNG repeats slipped out forming uneven bubbles that partly mimic insertion-deletion loops of mismatched DNA2. When the extruded hairpin exceeds 40-45 repeats, it can be bound by three or more MutSβ molecules, which are resistant to ATP-dependent dissociation. We envision that such MutSβ-CNG complexes recruit MutLγ endonuclease to nick DNA and initiate the repeat expansion process3,4. To develop drugs against the expansion diseases, we have identified lead compounds that prevent MutSβ binding to CNG repeats but not to mismatched DNA.
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Affiliation(s)
- Jun Li
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
| | - Huaibin Wang
- Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
| | - Wei Yang
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892
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19
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Medina-Rivera M, Phelps S, Sridharan M, Becker J, Lamb N, Kumar C, Sutton M, Bielinsky A, Balakrishnan L, Surtees J. Elevated MSH2 MSH3 expression interferes with DNA metabolism in vivo. Nucleic Acids Res 2023; 51:12185-12206. [PMID: 37930834 PMCID: PMC10711559 DOI: 10.1093/nar/gkad934] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 09/30/2023] [Accepted: 10/10/2023] [Indexed: 11/08/2023] Open
Abstract
The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.
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Affiliation(s)
- Melisa Medina-Rivera
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
| | - Samantha Phelps
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
| | - Madhumita Sridharan
- Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN, 46202, USA
| | - Jordan Becker
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Natalie A Lamb
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
| | - Charanya Kumar
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
| | - Mark D Sutton
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
| | - Anja Bielinsky
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Lata Balakrishnan
- Department of Biology, Indiana University Purdue University Indianapolis, Indianapolis, IN, 46202, USA
| | - Jennifer A Surtees
- Department of Biochemistry, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo NY, 14203, USA
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20
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Villy MC, Masliah-Planchon J, Schnitzler A, Delhomelle H, Buecher B, Filser M, Merchadou K, Golmard L, Melaabi S, Vacher S, Blanluet M, Suybeng V, Corsini C, Dhooge M, Hamzaoui N, Farelly S, Ait Omar A, Benamouzig R, Caumette V, Bahuau M, Cucherousset J, Allory Y, Stoppa-Lyonnet D, Bieche I, Colas C. MSH3: a confirmed predisposing gene for adenomatous polyposis. J Med Genet 2023; 60:1198-1205. [PMID: 37402566 DOI: 10.1136/jmg-2023-109341] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 06/18/2023] [Indexed: 07/06/2023]
Abstract
BACKGROUND The MSH3 gene is part of the DNA mismatch repair system, but has never been shown to be involved in Lynch syndrome. A first report of four patients from two families, bearing biallelic MSH3 germline variants, with a phenotype of attenuated colorectal adenomatous polyposis raised the question of its involvement in hereditary cancer predisposition. The patients' tumours exhibited elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), a hallmark of MSH3 deficiency. METHODS We report five new unrelated patients with MSH3-associated polyposis. We describe their personal and familial history and study the EMAST phenotype in various normal and tumour samples, which are relevant findings based on the rarity of this polyposis subtype so far. RESULTS All patients had attenuated colorectal adenomatous polyposis, with duodenal polyposis in two cases. Both women had breast carcinomas. EMAST phenotype was present at various levels in different samples of the five patients, confirming the MSH3 deficiency, with a gradient of instability in polyps depending on their degree of dysplasia. The negative EMAST phenotype ruled out the diagnosis of germline MSH3 deficiency for two patients: one homozygous for a benign variant and one with a monoallelic large deletion. CONCLUSION This report lends further credence to biallelic MSH3 germline pathogenic variants being involved in colorectal and duodenal adenomatous polyposis. Large-scale studies may help clarify the tumour spectrum and associated risks. Ascertainment of EMAST may help with the interpretation of variants of unknown significance. We recommend adding MSH3 to dedicated diagnostic gene panels.
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Affiliation(s)
| | | | - Anne Schnitzler
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Hélène Delhomelle
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Bruno Buecher
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Mathilde Filser
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | | | - Lisa Golmard
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Samia Melaabi
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Sophie Vacher
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Maud Blanluet
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Voreak Suybeng
- Department of Genetics, PSL University, Institut Curie, Paris, France
| | - Carole Corsini
- Medical Genetics Department, Centre Hospitalier Regional Universitaire de Montpellier, Montpellier, France
| | - Marion Dhooge
- Oncogenetic Unit, Department of Gastroenterology, AP-HP Centre-Université de Paris, Hopital Cochin, Paris, France
| | - Nadim Hamzaoui
- Department of Genetics, AP-HP Centre-Université de Paris, Hospital Cochin, Paris, France
| | - Solenne Farelly
- Oncogenetic Unit, Department of Gastroenterology, AP-HP Centre-Université de Paris, Hopital Cochin, Paris, France
| | - Amal Ait Omar
- Department of Gastroenterology, Hôpital Avicenne, Bobigny, France
| | | | - Vincent Caumette
- Department of Genetics, Hôpitaux Universitaires Henri Mondor, Creteil, France
| | - Michel Bahuau
- Department of Genetics, Hôpitaux Universitaires Henri Mondor, Creteil, France
| | - Joël Cucherousset
- Department of Pathology, GHI Le Raincy-Montfermeil, Montfermeil, France
| | - Yves Allory
- Department of Pathology, Université Paris-Saclay, Institut Curie, Paris, France
| | | | - Ivan Bieche
- Department of Genetics, Université Paris Cité, Institut Curie, Paris, France
| | - Chrystelle Colas
- Department of Genetics, PSL University, Institut Curie, Paris, France
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21
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Koeppel J, Weller J, Peets EM, Pallaseni A, Kuzmin I, Raudvere U, Peterson H, Liberante FG, Parts L. Prediction of prime editing insertion efficiencies using sequence features and DNA repair determinants. Nat Biotechnol 2023; 41:1446-1456. [PMID: 36797492 PMCID: PMC10567557 DOI: 10.1038/s41587-023-01678-y] [Citation(s) in RCA: 36] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 01/18/2023] [Indexed: 02/18/2023]
Abstract
Most short sequences can be precisely written into a selected genomic target using prime editing; however, it remains unclear what factors govern insertion. We design a library of 3,604 sequences of various lengths and measure the frequency of their insertion into four genomic sites in three human cell lines, using different prime editor systems in varying DNA repair contexts. We find that length, nucleotide composition and secondary structure of the insertion sequence all affect insertion rates. We also discover that the 3' flap nucleases TREX1 and TREX2 suppress the insertion of longer sequences. Combining the sequence and repair features into a machine learning model, we can predict relative frequency of insertions into a site with R = 0.70. Finally, we demonstrate how our accurate prediction and user-friendly software help choose codon variants of common fusion tags that insert at high efficiency, and provide a catalog of empirically determined insertion rates for over a hundred useful sequences.
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Affiliation(s)
| | | | | | | | - Ivan Kuzmin
- Department of Computer Science, University of Tartu, Tartu, Estonia
| | - Uku Raudvere
- Department of Computer Science, University of Tartu, Tartu, Estonia
| | - Hedi Peterson
- Department of Computer Science, University of Tartu, Tartu, Estonia
| | | | - Leopold Parts
- Wellcome Sanger Institute, Hinxton, UK.
- Department of Computer Science, University of Tartu, Tartu, Estonia.
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22
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Jayaraj A, Thayer KM, Beveridge DL, Hingorani MM. Molecular dynamics of mismatch detection-How MutS uses indirect readout to find errors in DNA. Biophys J 2023; 122:3031-3043. [PMID: 37329136 PMCID: PMC10432192 DOI: 10.1016/j.bpj.2023.06.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2022] [Revised: 04/30/2023] [Accepted: 06/12/2023] [Indexed: 06/18/2023] Open
Abstract
The mismatch repair protein MutS safeguards genomic integrity by finding and initiating repair of basepairing errors in DNA. Single-molecule studies show MutS diffusing on DNA, presumably scanning for mispaired/unpaired bases, and crystal structures show a characteristic "mismatch-recognition" complex with DNA enclosed within MutS and kinked at the site of error. But how MutS goes from scanning thousands of Watson-Crick basepairs to recognizing rare mismatches remains unanswered, largely because atomic-resolution data on the search process are lacking. Here, 10 μs all-atom molecular dynamics simulations of Thermus aquaticus MutS bound to homoduplex DNA and T-bulge DNA illuminate the structural dynamics underlying the search mechanism. MutS-DNA interactions constitute a multistep mechanism to check DNA over two helical turns for its 1) shape, through contacts with the sugar-phosphate backbone, 2) conformational flexibility, through bending/unbending engineered by large-scale motions of the clamp domain, and 3) local deformability, through basepair destabilizing contacts. Thus, MutS can localize a potential target by indirect readout due to lower energetic costs of bending mismatched DNA and identify a site that distorts easily due to weaker base stacking and pairing as a mismatch. The MutS signature Phe-X-Glu motif can then lock in the mismatch-recognition complex to initiate repair.
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Affiliation(s)
- Abhilash Jayaraj
- Chemistry Department, Wesleyan University, Middletown, Connecticut.
| | - Kelly M Thayer
- Chemistry Department, Wesleyan University, Middletown, Connecticut
| | | | - Manju M Hingorani
- Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut.
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23
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Zhao Z, Shang P, Mohanraju P, Geijsen N. Prime editing: advances and therapeutic applications. Trends Biotechnol 2023; 41:1000-1012. [PMID: 37002157 DOI: 10.1016/j.tibtech.2023.03.004] [Citation(s) in RCA: 72] [Impact Index Per Article: 36.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Revised: 02/24/2023] [Accepted: 03/03/2023] [Indexed: 04/01/2023]
Abstract
Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic and diagnostic landscape. However, CRISPR-Cas9, which edits DNA by activating DNA double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation repair is required. Prime editing, the latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without the requirement for DSBs. However, prime editing is still in its infancy, and further development is needed to improve editing efficiency and delivery strategies for therapeutic applications. We summarize latest developments in the optimization of prime editor (PE) variants with improved editing efficiency and precision. Moreover, we highlight some potential therapeutic applications.
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Affiliation(s)
- Zhihan Zhao
- Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands; The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden node, The Netherlands
| | - Peng Shang
- Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands; The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden node, The Netherlands
| | - Prarthana Mohanraju
- Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands; The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden node, The Netherlands.
| | - Niels Geijsen
- Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands; The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden node, The Netherlands.
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24
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Oh JM, Kang Y, Park J, Sung Y, Kim D, Seo Y, Lee E, Ra J, Amarsanaa E, Park YU, Lee S, Hwang J, Kim H, Schärer O, Cho S, Lee C, Takata KI, Lee J, Myung K. MSH2-MSH3 promotes DNA end resection during homologous recombination and blocks polymerase theta-mediated end-joining through interaction with SMARCAD1 and EXO1. Nucleic Acids Res 2023; 51:5584-5602. [PMID: 37140056 PMCID: PMC10287916 DOI: 10.1093/nar/gkad308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2022] [Revised: 04/04/2023] [Accepted: 04/27/2023] [Indexed: 05/05/2023] Open
Abstract
DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.
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Affiliation(s)
- Jung-Min Oh
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Oral Biochemistry, Dental and Life Science Institute, School of Dentistry, Pusan National University, Yangsan 50612, Republic of Korea
| | - Yujin Kang
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Jumi Park
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Yubin Sung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Dayoung Kim
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Yuri Seo
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Eun A Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Jae Sun Ra
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Enkhzul Amarsanaa
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Young-Un Park
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Seon Young Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Jung Me Hwang
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
| | - Hongtae Kim
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Orlando Schärer
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Seung Woo Cho
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Changwook Lee
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Kei-ichi Takata
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Ja Yil Lee
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biological Sciences, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
| | - Kyungjae Myung
- Center for Genomic Integrity, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
- Department of Biomedical Engineering, Ulsan National Institute of Science and Technology, Ulsan44919, Republic of Korea
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25
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Mätlik K, Baffuto M, Kus L, Deshmukh AL, Davis DA, Paul MR, Carroll TS, Caron MC, Masson JY, Pearson CE, Heintz N. Cell Type Specific CAG Repeat Expansions and Toxicity of Mutant Huntingtin in Human Striatum and Cerebellum. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.04.24.538082. [PMID: 37333326 PMCID: PMC10274669 DOI: 10.1101/2023.04.24.538082] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/20/2023]
Abstract
Brain region-specific degeneration and somatic expansions of the mutant Huntingtin (mHTT) CAG tract are key features of Huntington's disease (HD). However, the relationships between CAG expansions, death of specific cell types, and molecular events associated with these processes are not established. Here we employed fluorescence-activated nuclear sorting (FANS) and deep molecular profiling to gain insight into the properties of cell types of the human striatum and cerebellum in HD and control donors. CAG expansions arise in striatal medium spiny neurons (MSNs) and cholinergic interneurons, in cerebellar Purkinje neurons, and at mATXN3 in MSNs from SCA3 donors. CAG expansions in MSNs are associated with higher levels of MSH2 and MSH3 (forming MutSβ), which can inhibit nucleolytic excision of CAG slip-outs by FAN1 in a concentration-dependent manner. Our data indicate that ongoing CAG expansions are not sufficient for cell death, and identify transcriptional changes associated with somatic CAG expansions and striatal toxicity.
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26
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Park JC, Kim YJ, Han JH, Kim D, Park MJ, Kim J, Jang HK, Bae S, Cha HJ. MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells. MOLECULAR THERAPY. NUCLEIC ACIDS 2023; 32:914-922. [PMCID: PMC10280094 DOI: 10.1016/j.omtn.2023.05.015] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/04/2022] [Accepted: 05/10/2023] [Indexed: 06/22/2023]
Abstract
Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSβ determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSβ complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSβ by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSβ on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSβ in its outcome.
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Affiliation(s)
- Ju-Chan Park
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
| | - Yun-Jeong Kim
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
| | - Jun Hee Han
- Department of Chemistry, Hanyang University, Seoul, Republic of Korea
| | - Dayeon Kim
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
| | - Mihn Jeong Park
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
| | - Jumee Kim
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
| | - Hyeon-Ki Jang
- Division of Chemical Engineering and Bioengineering, College of Art Culture and Engineering, Kangwon National University, Chuncheon, South Korea
| | - Sangsu Bae
- College of Medicine, Seoul National University, Seoul, Republic of Korea
| | - Hyuk-Jin Cha
- College of Pharmacy, Seoul National University, Seoul, Republic of Korea
- Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea
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27
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Maksimov MO, Wu C, Ashbrook DG, Villani F, Colonna V, Mousavi N, Ma N, Lu L, Pritchard JK, Goren A, Williams RW, Palmer AA, Gymrek M. A novel quantitative trait locus implicates Msh3 in the propensity for genome-wide short tandem repeat expansions in mice. Genome Res 2023; 33:689-702. [PMID: 37127331 PMCID: PMC10317118 DOI: 10.1101/gr.277576.122] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Accepted: 04/26/2023] [Indexed: 05/03/2023]
Abstract
Short tandem repeats (STRs) are a class of rapidly mutating genetic elements typically characterized by repeated units of 1-6 bp. We leveraged whole-genome sequencing data for 152 recombinant inbred (RI) strains from the BXD family of mice to map loci that modulate genome-wide patterns of new mutations arising during parent-to-offspring transmission at STRs. We defined quantitative phenotypes describing the numbers and types of germline STR mutations in each strain and performed quantitative trait locus (QTL) analyses for each of these phenotypes. We identified a locus on Chromosome 13 at which strains inheriting the C57BL/6J (B) haplotype have a higher rate of STR expansions than those inheriting the DBA/2J (D) haplotype. The strongest candidate gene in this locus is Msh3, a known modifier of STR stability in cancer and at pathogenic repeat expansions in mice and humans, as well as a current drug target against Huntington's disease. The D haplotype at this locus harbors a cluster of variants near the 5' end of Msh3, including multiple missense variants near the DNA mismatch recognition domain. In contrast, the B haplotype contains a unique retrotransposon insertion. The rate of expansion covaries positively with Msh3 expression-with higher expression from the B haplotype. Finally, detailed analysis of mutation patterns showed that strains carrying the B allele have higher expansion rates, but slightly lower overall total mutation rates, compared with those with the D allele, particularly at tetranucleotide repeats. Our results suggest an important role for inherited variants in Msh3 in modulating genome-wide patterns of germline mutations at STRs.
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Affiliation(s)
- Mikhail O Maksimov
- Department of Medicine, University of California San Diego, La Jolla, California 92093, USA
- Department of Computer Science and Engineering, University of California San Diego, La Jolla, California 92093, USA
| | - Cynthia Wu
- Bioinformatics and Systems Biology Program, University of California San Diego, La Jolla, California 92093, USA
| | - David G Ashbrook
- Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
| | - Flavia Villani
- Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
| | - Vincenza Colonna
- Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
- Institute of Genetics and Biophysics, National Research Council, Naples 80111, Italy
| | - Nima Mousavi
- Department of Electrical and Computer Engineering, University of California San Diego, La Jolla, California 92093, USA
| | - Nichole Ma
- Department of Medicine, University of California San Diego, La Jolla, California 92093, USA
| | - Lu Lu
- Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
| | - Jonathan K Pritchard
- Department of Genetics, Stanford University, Stanford, California 94305, USA
- Department of Biology, Stanford University, Stanford, California 94305, USA
| | - Alon Goren
- Department of Medicine, University of California San Diego, La Jolla, California 92093, USA
- Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA
| | - Robert W Williams
- Department of Genetics, Genomics and Informatics, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA
| | - Abraham A Palmer
- Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA
- Department of Psychiatry, Department of Medicine, University of California San Diego, La Jolla, California 92093, USA
| | - Melissa Gymrek
- Department of Medicine, University of California San Diego, La Jolla, California 92093, USA;
- Department of Computer Science and Engineering, University of California San Diego, La Jolla, California 92093, USA
- Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA
- Department of Biomedical Informatics
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28
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Tam B, Qin Z, Zhao B, Wang SM, Lei CL. Integration of deep learning with Ramachandran plot molecular dynamics simulation for genetic variant classification. iScience 2023; 26:106122. [PMID: 36879825 PMCID: PMC9984559 DOI: 10.1016/j.isci.2023.106122] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2022] [Revised: 10/07/2022] [Accepted: 01/30/2023] [Indexed: 02/05/2023] Open
Abstract
Functional classification of genetic variants is a key for their clinical applications in patient care. However, abundant variant data generated by the next-generation DNA sequencing technologies limit the use of experimental methods for their classification. Here, we developed a protein structure and deep learning (DL)-based system for genetic variant classification, DL-RP-MDS, which comprises two principles: 1) Extracting protein structural and thermodynamics information using the Ramachandran plot-molecular dynamics simulation (RP-MDS) method, 2) combining those data with an unsupervised learning model of auto-encoder and a neural network classifier to identify the statistical significance patterns of the structural changes. We observed that DL-RP-MDS provided higher specificity than over 20 widely used in silico methods in classifying the variants of three DNA damage repair genes: TP53, MLH1, and MSH2. DL-RP-MDS offers a powerful platform for high-throughput genetic variant classification. The software and online application are available at https://genemutation.fhs.um.edu.mo/DL-RP-MDS/.
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Affiliation(s)
- Benjamin Tam
- Ministry of Education Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Zixin Qin
- Ministry of Education Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Bojin Zhao
- Ministry of Education Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - San Ming Wang
- Ministry of Education Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
| | - Chon Lok Lei
- Ministry of Education Frontiers Science Center for Precision Oncology, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.,Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Macau SAR, China
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29
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Abildgaard AB, Nielsen SV, Bernstein I, Stein A, Lindorff-Larsen K, Hartmann-Petersen R. Lynch syndrome, molecular mechanisms and variant classification. Br J Cancer 2023; 128:726-734. [PMID: 36434153 PMCID: PMC9978028 DOI: 10.1038/s41416-022-02059-z] [Citation(s) in RCA: 22] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Revised: 10/31/2022] [Accepted: 11/02/2022] [Indexed: 11/27/2022] Open
Abstract
Patients with the heritable cancer disease, Lynch syndrome, carry germline variants in the MLH1, MSH2, MSH6 and PMS2 genes, encoding the central components of the DNA mismatch repair system. Loss-of-function variants disrupt the DNA mismatch repair system and give rise to a detrimental increase in the cellular mutational burden and cancer development. The treatment prospects for Lynch syndrome rely heavily on early diagnosis; however, accurate diagnosis is inextricably linked to correct clinical interpretation of individual variants. Protein variant classification traditionally relies on cumulative information from occurrence in patients, as well as experimental testing of the individual variants. The complexity of variant classification is due to (1) that variants of unknown significance are rare in the population and phenotypic information on the specific variants is missing, and (2) that individual variant testing is challenging, costly and slow. Here, we summarise recent developments in high-throughput technologies and computational prediction tools for the assessment of variants of unknown significance in Lynch syndrome. These approaches may vastly increase the number of interpretable variants and could also provide important mechanistic insights into the disease. These insights may in turn pave the road towards developing personalised treatment approaches for Lynch syndrome.
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Affiliation(s)
- Amanda B Abildgaard
- The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - Sofie V Nielsen
- The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
| | - Inge Bernstein
- Department of Surgical Gastroenterology, Aalborg University Hospital, Aalborg, Denmark
- Institute of Clinical Medicine, Aalborg University Hospital, Aalborg University, Aalborg, Denmark
| | - Amelie Stein
- The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark
| | - Kresten Lindorff-Larsen
- The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
| | - Rasmus Hartmann-Petersen
- The Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Copenhagen, Denmark.
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30
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Bruekner SR, Pieters W, Fish A, Liaci AM, Scheffers S, Rayner E, Kaldenbach D, Drost L, Dekker M, van Hees-Stuivenberg S, Delzenne-Goette E, de Konink C, Houlleberghs H, Dubbink H, AlSaegh A, de Wind N, Förster F, te Riele H, Sixma T. Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding. Nucleic Acids Res 2023; 51:1173-1188. [PMID: 36715327 PMCID: PMC9943660 DOI: 10.1093/nar/gkad015] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2022] [Revised: 12/30/2022] [Accepted: 01/12/2023] [Indexed: 01/31/2023] Open
Abstract
The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.
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Affiliation(s)
| | | | - Alexander Fish
- Division of Biochemistry, Netherlands Cancer Institute and Oncode Institute, 1066 CX Amsterdam, The Netherlands
| | - A Manuel Liaci
- Structural Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584CH Utrecht, The Netherlands
| | - Serge Scheffers
- Division of Biochemistry, Netherlands Cancer Institute and Oncode Institute, 1066 CX Amsterdam, The Netherlands
| | - Emily Rayner
- Department of Human Genetics, Leiden University Medical Center, PO Box 9600 2300RC Leiden, The Netherlands
| | - Daphne Kaldenbach
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | - Lisa Drost
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | - Marleen Dekker
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | | | - Elly Delzenne-Goette
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | - Charlotte de Konink
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | - Hellen Houlleberghs
- Division of Tumor Biology and Immunology, Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
| | - Hendrikus Jan Dubbink
- Department of Pathology, Erasmus Medical Center, PO Box 2040 3000CA Rotterdam, The Netherlands
| | - Abeer AlSaegh
- Sultan Qaboos Comprehensive Cancer Care and Research Center, PO Box 787, 117 Muscat, Oman
| | - Niels de Wind
- Department of Human Genetics, Leiden University Medical Center, PO Box 9600 2300RC Leiden, The Netherlands
| | - Friedrich Förster
- Structural Biochemistry, Bijvoet Centre for Biomolecular Research, Utrecht University, 3584CH Utrecht, The Netherlands
| | - Hein te Riele
- Correspondence may also be addressed to Hein te Riele. Tel: +31 20 512 2084;
| | - Titia K Sixma
- To whom correspondence should be addressed: Tel: +31 20 512 1959;
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31
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Li X, Zhang G, Huang S, Liu Y, Tang J, Zhong M, Wang X, Sun W, Yao Y, Ji Q, Wang X, Liu J, Zhu S, Huang X. Development of a versatile nuclease prime editor with upgraded precision. Nat Commun 2023; 14:305. [PMID: 36658146 PMCID: PMC9852468 DOI: 10.1038/s41467-023-35870-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Accepted: 01/05/2023] [Indexed: 01/21/2023] Open
Abstract
The applicability of nuclease-based form of prime editor (PEn) has been hindered by its complexed editing outcomes. A chemical inhibitor against DNA-PK, which mediates the nonhomologous end joining (NHEJ) pathway, was recently shown to promote precise insertions by PEn. Nevertheless, the intrinsic issues of specificity and toxicity for such a chemical approach necessitate development of alternative strategies. Here, we find that co-introduction of PEn and a NHEJ-restraining, 53BP1-inhibitory ubiquitin variant potently drives precise edits via mitigation of unintended edits, framing a high-activity editing platform (uPEn) apparently complementing the canonical PE. Further developments involve exploring the effective configuration of a homologous region-containing pegRNA (HR-pegRNA). Overall, uPEn can empower high-efficiency installation of insertions (38%), deletions (43%) and replacements (52%) in HEK293T cells. When compared with PE3/5max, uPEn demonstrates superior activities for typically refractory base substitutions, and for small-block edits. Collectively, this work establishes a highly efficient PE platform with broad application potential.
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Affiliation(s)
- Xiangyang Li
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China.,Zhejiang Lab, Hangzhou, Zhejiang, 311121, China
| | - Guiquan Zhang
- Zhejiang Lab, Hangzhou, Zhejiang, 311121, China.,State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center at Medical School of Nanjing University, 210061, Nanjing, China
| | | | - Yao Liu
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China
| | - Jin Tang
- Zhejiang Lab, Hangzhou, Zhejiang, 311121, China
| | - Mingtian Zhong
- Institute for Brain Research and Rehabilitation, South China Normal University, Guangzhou, 510631, China
| | - Xin Wang
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China
| | - Wenjun Sun
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China
| | - Yuan Yao
- ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311215, China.,College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China
| | - Quanjiang Ji
- School of Physical Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China
| | - Xiaolong Wang
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, 712100, Yangling, Shaanxi, China
| | - Jianghuai Liu
- State Key Laboratory of Pharmaceutical Biotechnology, Model Animal Research Center at Medical School of Nanjing University, 210061, Nanjing, China.
| | - Shiqiang Zhu
- Zhejiang Lab, Hangzhou, Zhejiang, 311121, China.
| | - Xingxu Huang
- Gene Editing Center, School of Life Science and Technology, ShanghaiTech University, 100 Haike Rd., Pudong New Area, Shanghai, 201210, China. .,Zhejiang Lab, Hangzhou, Zhejiang, 311121, China.
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32
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Palmer N, Talib SZA, Goh CMF, Biswas K, Sharan SK, Kaldis P. Identification PMS1 and PMS2 as potential meiotic substrates of CDK2 activity. PLoS One 2023; 18:e0283590. [PMID: 36952545 PMCID: PMC10035876 DOI: 10.1371/journal.pone.0283590] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Accepted: 03/11/2023] [Indexed: 03/25/2023] Open
Abstract
Cyclin dependent-kinase 2 (CDK2) plays important functions during the mitotic cell cycle and also facilitates several key events during germ cell development. The majority of CDK2's known meiotic functions occur during prophase of the first meiotic division. Here, CDK2 is involved in the regulation of meiotic transcription, the pairing of homologous chromosomes, and the maturation of meiotic crossover sites. Despite that some of the CDK2 substrates are known, few of them display functions in meiosis. Here, we investigate potential meiotic CDK2 substrates using in silico and in vitro approaches. We find that CDK2 phosphorylates PMS2 at Thr337, PMS1 at Thr331, and MLH1 in vitro. Phosphorylation of PMS2 affects its interaction with MLH1 to some degree. In testis extracts from mice lacking Cdk2, there are changes in expression of PMS2, MSH2, and HEI10, which may be reflective of the loss of CDK2 phosphorylation. Our work has uncovered a few CDK2 substrates with meiotic functions, which will have to be verified in vivo. A better understanding of the CDK2 substrates will help us to gain deeper insight into the functions of this universal kinase.
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Affiliation(s)
- Nathan Palmer
- Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore
- Department of Chromosome Biology, Max Perutz Labs, University of Vienna, Vienna Biocenter, Vienna, Austria
| | - S Zakiah A Talib
- Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore
- Department Biologie II, Biozentrum der LMU München, Zell- und Entwicklungsbiologie, Planegg-Martinsried, Germany
| | - Christine M F Goh
- Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore
| | - Kajal Biswas
- Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, United States of America
| | - Shyam K Sharan
- Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, United States of America
| | - Philipp Kaldis
- Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore, Republic of Singapore
- Department of Clinical Sciences, Clinical Research Centre (CRC), Lund University, Malmö, Sweden
- Lund University Diabetes Centre, Lund University, Clinical Research Centre (CRC), Malmö, Sweden
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33
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Saber Sichani A, Ranjbar M, Baneshi M, Torabi Zadeh F, Fallahi J. A Review on Advanced CRISPR-Based Genome-Editing Tools: Base Editing and Prime Editing. Mol Biotechnol 2022; 65:849-860. [DOI: 10.1007/s12033-022-00639-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2022] [Accepted: 12/04/2022] [Indexed: 12/24/2022]
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34
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Ruprecht B, Wei L, Zheng L, Bodea S, Mo X, Maschberger M, Stoehr G, Hahne H, Cornella-Taracido I, Chi A. Chemoproteomic profiling to identify activity changes and functional inhibitors of DNA-binding proteins. Cell Chem Biol 2022; 29:1639-1648.e4. [PMID: 36356585 DOI: 10.1016/j.chembiol.2022.10.008] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2021] [Revised: 07/04/2022] [Accepted: 10/19/2022] [Indexed: 11/10/2022]
Abstract
DNA-binding proteins are promising therapeutic targets but are notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched subproteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound that displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific readout of transcription factor activity and can identify functional "hotspots" on DNA binders toward expanding the druggable proteome.
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Affiliation(s)
| | - Lan Wei
- Chemical Biology, Merck & Co., Inc, Boston, MA 02115, USA
| | - Li Zheng
- Chemical Biology, Merck & Co., Inc, Boston, MA 02115, USA
| | - Smaranda Bodea
- Chemical Biology, Merck & Co., Inc, Boston, MA 02115, USA
| | - Xuan Mo
- Chemical Biology, Merck & Co., Inc, Boston, MA 02115, USA
| | | | | | - Hannes Hahne
- OmicScouts GmbH, 85354 Freising, Bavaria, Germany
| | | | - An Chi
- Chemical Biology, Merck & Co., Inc, Boston, MA 02115, USA.
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35
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Rodent Models of Audiogenic Epilepsy: Genetic Aspects, Advantages, Current Problems and Perspectives. Biomedicines 2022; 10:biomedicines10112934. [PMID: 36428502 PMCID: PMC9687921 DOI: 10.3390/biomedicines10112934] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 11/10/2022] [Accepted: 11/11/2022] [Indexed: 11/18/2022] Open
Abstract
Animal models of epilepsy are of great importance in epileptology. They are used to study the mechanisms of epileptogenesis, and search for new genes and regulatory pathways involved in the development of epilepsy as well as screening new antiepileptic drugs. Today, many methods of modeling epilepsy in animals are used, including electroconvulsive, pharmacological in intact animals, and genetic, with the predisposition for spontaneous or refractory epileptic seizures. Due to the simplicity of manipulation and universality, genetic models of audiogenic epilepsy in rodents stand out among this diversity. We tried to combine data on the genetics of audiogenic epilepsy in rodents, the relevance of various models of audiogenic epilepsy to certain epileptic syndromes in humans, and the advantages of using of rodent strains predisposed to audiogenic epilepsy in current epileptology.
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36
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Armour-Garb I, Han ISM, Cowan BS, Thayer KM. Variable Regions of p53 Isoforms Allosterically Hard Code DNA Interaction. J Phys Chem B 2022; 126:8495-8507. [PMID: 36245142 PMCID: PMC9623584 DOI: 10.1021/acs.jpcb.2c06229] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
Allosteric regulation of protein activity pervades biology as the "second secret of life." We have been examining the allosteric regulation and mutant reactivation of the tumor suppressor protein p53. We have found that generalizing the definition of allosteric effector to include entire proteins and expanding the meaning of binding site to include the interface of a transcription factor with its DNA to be useful in understanding the modulation of protein activity. Here, we cast the variable regions of p53 isoforms as allosteric regulators of p53 interactions with its consensus DNA. We implemented molecular dynamics simulations and our lab's new techniques of molecular dynamics (MD) sectors and MD-Markov state models to investigate the effects of nine naturally occurring splice variant isoforms of p53. We find that all of the isoforms differ from wild type in their dynamic properties and how they interact with the DNA. We consider the implications of these findings on allostery and cancer treatment.
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Affiliation(s)
- Isabel Armour-Garb
- †Department
of Mathematics and Computer Science, ‡Department of Chemistry, and §College of Integrative
Sciences, Wesleyan University, Middletown, Connecticut 06457, United States
| | - In Sub Mark Han
- †Department
of Mathematics and Computer Science, ‡Department of Chemistry, and §College of Integrative
Sciences, Wesleyan University, Middletown, Connecticut 06457, United States
| | - Benjamin S. Cowan
- †Department
of Mathematics and Computer Science, ‡Department of Chemistry, and §College of Integrative
Sciences, Wesleyan University, Middletown, Connecticut 06457, United States
| | - Kelly M. Thayer
- †Department
of Mathematics and Computer Science, ‡Department of Chemistry, and §College of Integrative
Sciences, Wesleyan University, Middletown, Connecticut 06457, United States,
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37
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DuPrie ML, Palacio T, Calil FA, Kolodner RD, Putnam CD. Mlh1 interacts with both Msh2 and Msh6 for recruitment during mismatch repair. DNA Repair (Amst) 2022; 119:103405. [PMID: 36122480 PMCID: PMC9639671 DOI: 10.1016/j.dnarep.2022.103405] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2022] [Revised: 08/30/2022] [Accepted: 09/10/2022] [Indexed: 11/29/2022]
Abstract
Eukaryotic DNA mismatch repair (MMR) initiates through mispair recognition by the MutS homologs Msh2-Msh6 and Msh2-Msh3 and subsequent recruitment of the MutL homologs Mlh1-Pms1 (human MLH1-PMS2). In bacteria, MutL is recruited by interactions with the connector domain of one MutS subunit and the ATPase and core domains of the other MutS subunit. Analysis of the S. cerevisiae and human homologs have only identified an interaction between the Msh2 connector domain and Mlh1. Here we investigated whether a conserved Msh6 ATPase/core domain-Mlh1 interaction and an Msh2-Msh6 interaction with Pms1 also act in MMR. Mutations in MLH1 affecting interactions with both the Msh2 and Msh6 interfaces caused MMR defects, whereas equivalent pms1 mutations did not cause MMR defects. Mutant Mlh1-Pms1 complexes containing Mlh1 amino acid substitutions were defective for recruitment to mispaired DNA by Msh2-Msh6, did not support MMR in reconstituted Mlh1-Pms1-dependent MMR reactions in vitro, but were proficient in Msh2-Msh6-independent Mlh1-Pms1 endonuclease activity. These results indicate that Mlh1, the common subunit of the Mlh1-Pms1, Mlh1-Mlh2, and Mlh1-Mlh3 complexes, but not Pms1, is recruited by Msh2-Msh6 through interactions with both of its subunits.
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Affiliation(s)
- Matthew L DuPrie
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA
| | - Tatiana Palacio
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA
| | - Felipe A Calil
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA
| | - Richard D Kolodner
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA; Department of Cellular and Molecular Medicine University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA; Moores-UCSD Cancer Center University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA; Institute of Genomic Medicine University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA
| | - Christopher D Putnam
- Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA; Department of Medicine University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0660, USA.
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38
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Liu N, Zhou L, Lin G, Hu Y, Jiao Y, Wang Y, Liu J, Yang S, Yao S. HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 29:36-46. [PMID: 35784015 PMCID: PMC9207553 DOI: 10.1016/j.omtn.2022.05.036] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 09/19/2021] [Accepted: 05/27/2022] [Indexed: 01/19/2023]
Abstract
Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.
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Affiliation(s)
- Nan Liu
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Lifang Zhou
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Guifeng Lin
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Yun Hu
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Yaoge Jiao
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Yanhong Wang
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Jingming Liu
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Shengyong Yang
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
| | - Shaohua Yao
- Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu 610041, Sichuan, China
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Abstract
Roughly 3% of the human genome consists of microsatellites or tracts of short tandem repeats (STRs). These STRs are often unstable, undergoing high-frequency expansions (increases) or contractions (decreases) in the number of repeat units. Some microsatellite instability (MSI) is seen at multiple STRs within a single cell and is associated with certain types of cancer. A second form of MSI is characterised by expansion of a single gene-specific STR and such expansions are responsible for a group of 40+ human genetic disorders known as the repeat expansion diseases (REDs). While the mismatch repair (MMR) pathway prevents genome-wide MSI, emerging evidence suggests that some MMR factors are directly involved in generating expansions in the REDs. Thus, MMR suppresses some forms of expansion while some MMR factors promote expansion in other contexts. This review will cover what is known about the paradoxical effect of MMR on microsatellite expansion in mammalian cells.
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40
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Kabbage M, Ben Aissa-Haj J, Othman H, Jaballah-Gabteni A, Laarayedh S, Elouej S, Medhioub M, Kettiti HT, Khsiba A, Mahmoudi M, BelFekih H, Maaloul A, Touinsi H, Hamzaoui L, Chelbi E, Abdelhak S, Boubaker MS, Azzouz MM. A Rare MSH2 Variant as a Candidate Marker for Lynch Syndrome II Screening in Tunisia: A Case of Diffuse Gastric Carcinoma. Genes (Basel) 2022; 13:genes13081355. [PMID: 36011265 PMCID: PMC9407052 DOI: 10.3390/genes13081355] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/17/2022] [Accepted: 07/21/2022] [Indexed: 12/24/2022] Open
Abstract
Several syndromic forms of digestive cancers are known to predispose to early-onset gastric tumors such as Hereditary Diffuse Gastric Cancer (HDGC) and Lynch Syndrome (LS). LSII is an extracolonic cancer syndrome characterized by a tumor spectrum including gastric cancer (GC). In the current work, our main aim was to identify the mutational spectrum underlying the genetic predisposition to diffuse gastric tumors occurring in a Tunisian family suspected of both HDGC and LS II syndromes. We selected the index case “JI-021”, which was a woman diagnosed with a Diffuse Gastric Carcinoma and fulfilling the international guidelines for both HDGC and LSII syndromes. For DNA repair, a custom panel targeting 87 candidate genes recovering the four DNA repair pathways was used. Structural bioinformatics analysis was conducted to predict the effect of the revealed variants on the functional properties of the proteins. DNA repair genes panel screening identified two variants: a rare MSH2 c.728G>A classified as a variant with uncertain significance (VUS) and a novel FANCD2 variant c.1879G>T. The structural prediction model of the MSH2 variant and electrostatic potential calculation showed for the first time that MSH2 c.728G>A is likely pathogenic and is involved in the MSH2-MLH1 complex stability. It appears to affect the MSH2-MLH1 complex as well as DNA-complex stability. The c.1879G>T FANCD2 variant was predicted to destabilize the protein structure. Our results showed that the MSH2 p.R243Q variant is likely pathogenic and is involved in the MSH2-MLH1 complex stability, and molecular modeling analysis highlights a putative impact on the binding with MLH1 by disrupting the electrostatic potential, suggesting the revision of its status from VUS to likely pathogenic. This variant seems to be a shared variant in the Mediterranean region. These findings emphasize the importance of testing DNA repair genes for patients diagnosed with diffuse GC with suspicion of LSII and colorectal cancer allowing better clinical surveillance for more personalized medicine.
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Affiliation(s)
- Maria Kabbage
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Correspondence:
| | - Jihenne Ben Aissa-Haj
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Houcemeddine Othman
- Sydney Brenner Institute for Molecular Bioscience, University of the Witwatersrand, Johannesburg 2000, South Africa;
| | - Amira Jaballah-Gabteni
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Sarra Laarayedh
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Sahar Elouej
- Marseille Medical Genetics, Aix Marseille University, INSERM, 13007 Marseille, France;
| | - Mouna Medhioub
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Gastroenterology Department, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Haifa Tounsi Kettiti
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Amal Khsiba
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Gastroenterology Department, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Moufida Mahmoudi
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Gastroenterology Department, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Houda BelFekih
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Department of Oncology, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Afifa Maaloul
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
| | - Hassen Touinsi
- Department of Surgery, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia;
| | - Lamine Hamzaoui
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Gastroenterology Department, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Emna Chelbi
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Department of Pathology, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
| | - Sonia Abdelhak
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Mohamed Samir Boubaker
- Department of Human and Experimental Pathology, Institut Pasteur de Tunis, Tunis 1002, Tunisia; (J.B.A.-H.); (A.J.-G.); (S.L.); (H.T.K.); (A.M.); (M.S.B.)
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
| | - Mohamed Mousaddak Azzouz
- Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunis EL Manar University, Tunis 1002, Tunisia; (M.M.); (A.K.); (M.M.); (H.B.); (L.H.); (E.C.); (S.A.); (M.M.A.)
- Gastroenterology Department, Mohamed Tahar Maamouri Hospital, Nabeul 8000, Tunisia
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41
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Sakellariou D, Bak ST, Isik E, Barroso SI, Porro A, Aguilera A, Bartek J, Janscak P, Peña-Diaz J. MutSβ regulates G4-associated telomeric R-loops to maintain telomere integrity in ALT cancer cells. Cell Rep 2022; 39:110602. [PMID: 35385755 DOI: 10.1016/j.celrep.2022.110602] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2021] [Revised: 12/17/2021] [Accepted: 03/10/2022] [Indexed: 12/24/2022] Open
Abstract
Up to 15% of human cancers maintain their telomeres through a telomerase-independent mechanism, termed "alternative lengthening of telomeres" (ALT) that relies on homologous recombination between telomeric sequences. Emerging evidence suggests that the recombinogenic nature of ALT telomeres results from the formation of RNA:DNA hybrids (R-loops) between telomeric DNA and the long-noncoding telomeric repeat-containing RNA (TERRA). Here, we show that the mismatch repair protein MutSβ, a heterodimer of MSH2 and MSH3 subunits, is enriched at telomeres in ALT cancer cells, where it prevents the accumulation of telomeric G-quadruplex (G4) structures and R-loops. Cells depleted of MSH3 display increased incidence of R-loop-dependent telomere fragility and accumulation of telomeric C-circles. We also demonstrate that purified MutSβ recognizes and destabilizes G4 structures in vitro. These data suggest that MutSβ destabilizes G4 structures in ALT telomeres to regulate TERRA R-loops, which is a prerequisite for maintenance of telomere integrity during ALT.
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Affiliation(s)
- Despoina Sakellariou
- Center for Healthy Aging, Department of Neuroscience and Pharmacology, University of Copenhagen, 2200 Copenhagen, Denmark; Danish Cancer Society Research Center, 2100 Copenhagen, Denmark
| | - Sara Thornby Bak
- Center for Healthy Aging, Department of Neuroscience and Pharmacology, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Esin Isik
- Institute of Molecular Cancer Research, University of Zurich, 8057 Zürich, Switzerland
| | - Sonia I Barroso
- Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, University of Seville-CSIC-UPO, Seville, Spain
| | - Antonio Porro
- Institute of Molecular Cancer Research, University of Zurich, 8057 Zürich, Switzerland
| | - Andrés Aguilera
- Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, University of Seville-CSIC-UPO, Seville, Spain
| | - Jiri Bartek
- Danish Cancer Society Research Center, 2100 Copenhagen, Denmark; Division of Genome Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institute, 17177 Stockholm, Sweden; Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 14300 Prague, Czech Republic
| | - Pavel Janscak
- Institute of Molecular Cancer Research, University of Zurich, 8057 Zürich, Switzerland; Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, 14300 Prague, Czech Republic.
| | - Javier Peña-Diaz
- Center for Healthy Aging, Department of Neuroscience and Pharmacology, University of Copenhagen, 2200 Copenhagen, Denmark.
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42
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Fukui K, Inoue M, Murakawa T, Baba S, Kumasaka T, Yano T. Structural and functional insights into the mechanism by which MutS2 recognizes a DNA junction. Structure 2022; 30:973-982.e4. [DOI: 10.1016/j.str.2022.03.014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2022] [Revised: 03/11/2022] [Accepted: 03/23/2022] [Indexed: 10/18/2022]
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43
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Brovarets’ OO, Muradova A, Hovorun DM. Novel horizons of the conformationally-tautomeric transformations of the G·T base pairs: quantum-mechanical investigation. Mol Phys 2022. [DOI: 10.1080/00268976.2022.2026510] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Affiliation(s)
- Ol’ha O. Brovarets’
- Department of Molecular and Quantum Biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine
| | - Alona Muradova
- Department of Molecular Biotechnology and Bioinformatics, Institute of High Technologies, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
| | - Dmytro M. Hovorun
- Department of Molecular and Quantum Biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv, Ukraine
- Department of Molecular Biotechnology and Bioinformatics, Institute of High Technologies, Taras Shevchenko National University of Kyiv, Kyiv, Ukraine
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44
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Charbonnier JB. Tandem regulation of MutS activity by ATP and DNA during MMR initiation. Nat Struct Mol Biol 2022; 29:5-7. [PMID: 35027743 DOI: 10.1038/s41594-021-00713-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Jean Baptiste Charbonnier
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Gif-sur-Yvette, France.
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45
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Barroso-González J, García-Expósito L, Galaviz P, Lynskey ML, Allen JAM, Hoang S, Watkins SC, Pickett HA, O'Sullivan RJ. Anti-recombination function of MutSα restricts telomere extension by ALT-associated homology-directed repair. Cell Rep 2021; 37:110088. [PMID: 34879271 PMCID: PMC8724847 DOI: 10.1016/j.celrep.2021.110088] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 09/13/2021] [Accepted: 11/10/2021] [Indexed: 01/02/2023] Open
Abstract
Alternative lengthening of telomeres (ALT) is a telomere-elongation mechanism observed in ~15% of cancer subtypes. Current models indicate that ALT is mediated by homology-directed repair mechanisms. By disrupting MSH6 gene expression, we show that the deficiency of MutSα (MSH2/MSH6) DNA mismatch repair complex causes striking telomere hyperextension. Mechanistically, we show MutSα is specifically recruited to telomeres in ALT cells by associating with the proliferating-cell nuclear antigen (PCNA) subunit of the ALT telomere replisome. We also provide evidence that MutSα counteracts Bloom (BLM) helicase, which adopts a crucial role in stabilizing hyper-extended telomeres and maintaining the survival of MutSα-deficient ALT cancer cells. Lastly, we propose a model in which MutSα deficiency impairs heteroduplex rejection, leading to premature initiation of telomere DNA synthesis that coincides with an accumulation of telomere variant repeats (TVRs). These findings provide evidence that the MutSα DNA mismatch repair complex acts to restrain unwarranted ALT. Barroso-Gonzalez et al. show that the mismatch repair complex MutSα restricts the alternative lengthening of telomeres (ALT) pathway in cancer cells. MutSα has an anti-recombination function and limits recombination between heteroduplex sequences at telomeres, in part by counteracting the Bloom helicase (BLM).
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Affiliation(s)
- Jonathan Barroso-González
- Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
| | - Laura García-Expósito
- Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
| | - Pablo Galaviz
- Bioinformatics Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia
| | - Michelle Lee Lynskey
- Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
| | - Joshua A M Allen
- Telomere Length Regulation Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia
| | - SongMy Hoang
- Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
| | - Simon C Watkins
- Department of Cell Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
| | - Hilda A Pickett
- Telomere Length Regulation Unit, Children's Medical Research Institute, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia
| | - Roderick J O'Sullivan
- Department of Pharmacology and Chemical Biology, School of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
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46
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Chuvakova LN, Funikov SY, Rezvykh AP, Davletshin AI, Evgen'ev MB, Litvinova SA, Fedotova IB, Poletaeva II, Garbuz DG. Transcriptome of the Krushinsky-Molodkina Audiogenic Rat Strain and Identification of Possible Audiogenic Epilepsy-Associated Genes. Front Mol Neurosci 2021; 14:738930. [PMID: 34803604 PMCID: PMC8600260 DOI: 10.3389/fnmol.2021.738930] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 10/07/2021] [Indexed: 12/13/2022] Open
Abstract
Audiogenic epilepsy (AE), inherent to several rodent strains is widely studied as a model of generalized convulsive epilepsy. The molecular mechanisms that determine the manifestation of AE are not well understood. In the present work, we compared transcriptomes from the corpora quadrigemina in the midbrain zone, which are crucial for AE development, to identify genes associated with the AE phenotype. Three rat strains without sound exposure were compared: Krushinsky-Molodkina (KM) strain (100% AE-prone); Wistar outbred rat strain (non-AE prone) and “0” strain (partially AE-prone), selected from F2 KM × Wistar hybrids for their lack of AE. The findings showed that the KM strain gene expression profile exhibited a number of characteristics that differed from those of the Wistar and “0” strain profiles. In particular, the KM rats showed increased expression of a number of genes involved in the positive regulation of the MAPK signaling cascade and genes involved in the positive regulation of apoptotic processes. Another characteristic of the KM strain which differed from that of the Wistar and “0” rats was a multi-fold increase in the expression level of the Ttr gene and a significant decrease in the expression of the Msh3 gene. Decreased expression of a number of oxidative phosphorylation-related genes and a few other genes was also identified in the KM strain. Our data confirm the complex multigenic nature of AE inheritance in rodents. A comparison with data obtained from other independently selected AE-prone rodent strains suggests some common causes for the formation of the audiogenic phenotype.
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Affiliation(s)
- Lyubov N Chuvakova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Sergei Yu Funikov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Alexander P Rezvykh
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia.,Moscow Institute of Physics and Technology, Dolgoprudny, Russia
| | - Artem I Davletshin
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | - Michael B Evgen'ev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
| | | | | | | | - David G Garbuz
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
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Abstract
Mismatched base pairs alter the flexibility and intrinsic curvature of DNA. The role of such DNA features is not fully understood in the mismatch repair pathway. MutS/DNA complexes exhibit DNA bending, PHE intercalation, and changes of base-pair parameters near the mismatch. Recently, we have shown that base-pair opening in the absence of MutS can discriminate mismatches from canonical base pairs better than DNA bending. However, DNA bending in the absence of MutS was found to be rather challenging to describe correctly. Here, we present a computational study on the DNA bending of canonical and G/T mismatched DNAs. Five types of geometric parameters covering template-based bending toward the experimental DNA structure, global, and local geometry parameters were employed in biased molecular dynamics in the absence of MutS. None of these parameters showed higher discrimination than the base-pair opening. Only roll could induce a sharply localized bending of DNA as observed in the experimental MutS/DNA structure. Further, we demonstrated that the intercalation of benzene mimicking PHE decreases the energetic cost of DNA bending without any effect on mismatch discrimination.
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Affiliation(s)
- Tomáš Bouchal
- National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.,CEITEC─Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
| | - Ivo Durník
- National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.,CEITEC─Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
| | - Petr Kulhánek
- National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.,CEITEC─Central European Institute of Technology, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
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48
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Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, Chen C, Nelson JW, Newby GA, Sahin M, Osborn MJ, Weissman JS, Adamson B, Liu DR. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 2021; 184:5635-5652.e29. [PMID: 34653350 PMCID: PMC8584034 DOI: 10.1016/j.cell.2021.09.018] [Citation(s) in RCA: 430] [Impact Index Per Article: 107.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2021] [Revised: 08/09/2021] [Accepted: 09/09/2021] [Indexed: 12/26/2022]
Abstract
While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.
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Affiliation(s)
- Peter J Chen
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
| | - Jeffrey A Hussmann
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Jun Yan
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
| | - Friederike Knipping
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55454, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55108, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Purnima Ravisankar
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA
| | - Pin-Fang Chen
- Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA
| | - Cidi Chen
- Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA
| | - James W Nelson
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
| | - Gregory A Newby
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA
| | - Mustafa Sahin
- Human Neuron Core, Rosamund Stone Zander Translational Neuroscience Center, Boston Children's Hospital, Boston, MA 02115, USA; Department of Neurology, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA
| | - Mark J Osborn
- Department of Pediatrics, University of Minnesota, Minneapolis, MN 55454, USA; Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55108, USA; Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA
| | - Jonathan S Weissman
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA
| | - Britt Adamson
- Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544, USA.
| | - David R Liu
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02141, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
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49
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Muthye V, Lavrov DV. Multiple Losses of MSH1, Gain of mtMutS, and Other Changes in the MutS Family of DNA Repair Proteins in Animals. Genome Biol Evol 2021; 13:evab191. [PMID: 34402879 PMCID: PMC8438181 DOI: 10.1093/gbe/evab191] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/11/2021] [Indexed: 12/15/2022] Open
Abstract
MutS is a key component of the mismatch repair (MMR) pathway. Members of the MutS protein family are present in prokaryotes, eukaryotes, and viruses. Six MutS homologs (MSH1-6) have been identified in yeast, of which three function in nuclear MMR, while MSH1 functions in mitochondrial DNA repair. MSH proteins are believed to be well conserved in animals, except for MSH1-which is thought to be lost. Two intriguing exceptions to this general picture have been found, both in the class Anthozoa within the phylum Cnidaria. First, an ortholog of the yeast-MSH1 was reported in one hexacoral species. Second, a MutS homolog (mtMutS) has been found in the mitochondrial genome of all octocorals. To understand the origin and potential functional implications of these exceptions, we investigated the evolution of the MutS family both in Cnidaria and in animals in general. Our study confirmed the acquisition of octocoral mtMutS by horizontal gene transfer from a giant virus. Surprisingly, we identified MSH1 in all hexacorals and several sponges and placozoans. By contrast, MSH1 orthologs were lacking in other cnidarians, ctenophores, and bilaterian animals. Furthermore, while we identified MSH2 and MSH6 in nearly all animals, MSH4, MSH5, and, especially, MSH3 were missing in multiple species. Overall, our analysis revealed a dynamic evolution of the MutS family in animals, with multiple losses of MSH1, MSH3, some losses of MSH4 and MSH5, and a gain of the octocoral mtMutS. We propose that octocoral mtMutS functionally replaced MSH1 that was present in the common ancestor of Anthozoa.
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Affiliation(s)
- Viraj Muthye
- Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa
| | - Dennis V Lavrov
- Department of Ecology, Evolution and Organismal Biology, Iowa State University, Ames, Iowa
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50
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Elez M. Mismatch Repair: From Preserving Genome Stability to Enabling Mutation Studies in Real-Time Single Cells. Cells 2021; 10:cells10061535. [PMID: 34207040 PMCID: PMC8235422 DOI: 10.3390/cells10061535] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 06/11/2021] [Accepted: 06/15/2021] [Indexed: 12/18/2022] Open
Abstract
Mismatch Repair (MMR) is an important and conserved keeper of the maintenance of genetic information. Miroslav Radman's contributions to the field of MMR are multiple and tremendous. One of the most notable was to provide, along with Bob Wagner and Matthew Meselson, the first direct evidence for the existence of the methyl-directed MMR. The purpose of this review is to outline several aspects and biological implications of MMR that his work has helped unveil, including the role of MMR during replication and recombination editing, and the current understanding of its mechanism. The review also summarizes recent discoveries related to the visualization of MMR components and discusses how it has helped shape our understanding of the coupling of mismatch recognition to replication. Finally, the author explains how visualization of MMR components has paved the way to the study of spontaneous mutations in living cells in real time.
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Affiliation(s)
- Marina Elez
- Micalis Institute, INRAE, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France;
- Laboratoire Jean Perrin (LJP), Institut de Biologie Paris-Seine (IBPS), CNRS, Sorbonne Université, F-75005 Paris, France
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