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Guevara-Ramírez P, Ruiz-Pozo VA, Cadena-Ullauri S, Paz-Cruz E, Tamayo-Trujillo R, Gaviria A, Cevallos F, Zambrano AK. Case report: Exploring Lynch Syndrome through genomic analysis in a mestizo Ecuadorian patient and his brother. Front Med (Lausanne) 2024; 11:1498290. [PMID: 39741510 PMCID: PMC11685006 DOI: 10.3389/fmed.2024.1498290] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2024] [Accepted: 11/22/2024] [Indexed: 01/03/2025] Open
Abstract
Lynch Syndrome (LS) is a hereditary disorder characterized by genetic mutations in DNA mismatch repair genes, affecting approximately 0.35% of the population. LS primarily increases the risk of colorectal cancer (CRC), as well as various other cancer types like endometrial, breast, and gastric cancers. Microsatellite instability, caused by MMR gene mutations, is a key feature of LS, impacting genes such as MLH1, MSH2, MSH6, and PMS2. Pathology tests studying microsatellite instability and immunohistochemical staining are used to diagnose LS. Furthermore, next-generation sequencing (NGS) allows for a thorough investigation of cancer susceptibility genes. This approach is crucial for identifying affected individuals and managing their care effectively. This study evaluated two siblings who harbored a mutation in the MLH1 gene associated with LS. The older brother was diagnosed with CRC at 24, while the younger brother remains asymptomatic at 7 years old. Genetic testing confirmed the presence of the MLH1 mutation in both siblings. Ancestry analysis showed a mix of African, European, and Native American heritage, common among Ecuadorians. Both siblings shared a family history of cancer, suggesting hereditary factors. Treatment involved surgery and chemotherapy for the older brother, emphasizing the importance of genetic testing for siblings with a cancer family history. NGS plays a pivotal role in identifying genetic mutations and guiding treatment decisions, demonstrating its significance in managing LS and other hereditary cancers.
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Affiliation(s)
- Patricia Guevara-Ramírez
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
| | - Viviana A. Ruiz-Pozo
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
| | - Santiago Cadena-Ullauri
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
| | - Elius Paz-Cruz
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
| | - Rafael Tamayo-Trujillo
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
| | - Aníbal Gaviria
- Hemocentro Nacional, Cruz Roja Ecuatoriana, Quito, Ecuador
| | | | - Ana Karina Zambrano
- Centro de Investigación Genética y Genómica, Facultad de Ciencias de la Salud Eugenio Espejo, Universidad UTE, Quito, Ecuador
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Chatterjee T, Tiwari A, Gupta R, Shukla H, Varshney A, Mishra S, Habib S. A Plasmodium apicoplast-targeted unique exonuclease/FEN exhibits interspecies functional differences attributable to an insertion that alters DNA-binding. Nucleic Acids Res 2024; 52:7843-7862. [PMID: 38888125 PMCID: PMC11260460 DOI: 10.1093/nar/gkae512] [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: 07/25/2023] [Revised: 05/30/2024] [Accepted: 06/03/2024] [Indexed: 06/20/2024] Open
Abstract
The human malaria parasite Plasmodium falciparum genome is among the most A + T rich, with low complexity regions (LCRs) inserted in coding sequences including those for proteins targeted to its essential relict plastid (apicoplast). Replication of the apicoplast genome (plDNA), mediated by the atypical multifunctional DNA polymerase PfPrex, would require additional enzymatic functions for lagging strand processing. We identified an apicoplast-targeted, [4Fe-4S]-containing, FEN/Exo (PfExo) with a long LCR insertion and detected its interaction with PfPrex. Distinct from other known exonucleases across organisms, PfExo recognized a wide substrate range; it hydrolyzed 5'-flaps, processed dsDNA as a 5'-3' exonuclease, and was a bipolar nuclease on ssDNA and RNA-DNA hybrids. Comparison with the rodent P. berghei ortholog PbExo, which lacked the insertion and [4Fe-4S], revealed interspecies functional differences. The insertion-deleted PfExoΔins behaved like PbExo with a limited substrate repertoire because of compromised DNA binding. Introduction of the PfExo insertion into PbExo led to gain of activities that the latter initially lacked. Knockout of PbExo indicated essentiality of the enzyme for survival. Our results demonstrate the presence of a novel apicoplast exonuclease with a functional LCR that diversifies substrate recognition, and identify it as the candidate flap-endonuclease and RNaseH required for plDNA replication and maintenance.
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Affiliation(s)
- Tribeni Chatterjee
- Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Anupama Tiwari
- Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Ritika Gupta
- Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Himadri Shukla
- Division of Molecular Microbiology and Immunology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Aastha Varshney
- Division of Molecular Microbiology and Immunology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Satish Mishra
- Division of Molecular Microbiology and Immunology, CSIR-Central Drug Research Institute, Lucknow, India
| | - Saman Habib
- Division of Biochemistry and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India
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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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Umar AA, Liddell S, Hussain R, Siligardi G, Harris G, Carr S, Asiani K, Gowers DM, Odell M, Scott DJ. Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation. Biochim Biophys Acta Gen Subj 2020; 1864:129730. [PMID: 32926959 DOI: 10.1016/j.bbagen.2020.129730] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2020] [Revised: 08/21/2020] [Accepted: 09/08/2020] [Indexed: 12/19/2022]
Abstract
BACKGROUND Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3'-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. METHODS Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. RESULTS AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed β-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14-3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. CONCLUSIONS This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection. SIGNIFICANCE The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14-3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14-3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy.
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Affiliation(s)
- Aminu Argungu Umar
- School of Biosciences, University of Nottingham Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom; Department of Biochemistry, Kebbi State University of Science and Technology, Aliero, P.M.B 1144, Birnin Kebbi, Nigeria.
| | - Susan Liddell
- School of Biosciences, University of Nottingham Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom
| | - Rohanah Hussain
- Diamond Light Source, Rutherford Appleton Laboratory, Oxfordshire OX11 0DE, United Kingdom
| | - Giuliano Siligardi
- Diamond Light Source, Rutherford Appleton Laboratory, Oxfordshire OX11 0DE, United Kingdom
| | - Gemma Harris
- Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire OX11 0FA, United Kingdom
| | - Stephen Carr
- Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire OX11 0FA, United Kingdom
| | - Karishma Asiani
- School of Biosciences, University of Nottingham Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom
| | - Darren M Gowers
- School of Biological Science, King Henry Building, King Henry 1(st) Street, Portsmouth, Hampshire PO1 2DY, United Kingdom
| | - Mark Odell
- Department of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, United Kingdom
| | - David J Scott
- School of Biosciences, University of Nottingham Sutton Bonington Campus, Leicestershire LE12 5RD, United Kingdom; Research Complex at Harwell, Rutherford Appleton Laboratory, Oxfordshire OX11 0FA, United Kingdom; ISIS Spallation Neutron and Muon source, Rutherford Appleton Laboratory, Oxfordshire OX11 0QX, United Kingdom
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Tamura K, Kaneda M, Futagawa M, Takeshita M, Kim S, Nakama M, Kawashita N, Tatsumi-Miyajima J. Genetic and genomic basis of the mismatch repair system involved in Lynch syndrome. Int J Clin Oncol 2019; 24:999-1011. [PMID: 31273487 DOI: 10.1007/s10147-019-01494-y] [Citation(s) in RCA: 45] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Accepted: 06/17/2019] [Indexed: 12/11/2022]
Abstract
Lynch syndrome is a cancer-predisposing syndrome inherited in an autosomal-dominant manner, wherein colon cancer and endometrial cancer develop frequently in the family, it results from a loss-of-function mutation in one of four different genes (MLH1, MSH2, MSH6, and PMS2) encoding mismatch repair proteins. Being located immediately upstream of the MSH2 gene, EPCAM abnormalities can affect MSH2 and cause Lynch syndrome. Mismatch repair proteins are involved in repairing of incorrect pairing (point mutations and deletion/insertion of simple repetitive sequences, so-called microsatellites) that can arise during DNA replication. MSH2 forms heterodimers with MSH6 or MSH3 (MutSα, MutSβ, respectively) and is involved in mismatch-pair recognition and initiation of repair. MLH1 forms a complex with PMS2, and functions as an endonuclease. If the mismatch repair system is thoroughly working, genome integrity is maintained completely. Lynch syndrome is a state of mismatch repair deficiency due to a monoallelic abnormality of any mismatch repair genes. The phenotype indicating the mismatch repair deficiency can be frequently shown as a microsatellite instability in tumors. Children with germline biallelic mismatch repair gene abnormalities were reported to develop conditions such as gastrointestinal polyposis, colorectal cancer, brain cancer, leukemia, etc., and so on, demonstrating the need to respond with new concepts in genetic counseling. In promoting cancer genome medicine in a new era, such as by utilizing immune checkpoints, it is important to understand the genetic and genomic molecular background, including the status of mismatch repair deficiency.
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Affiliation(s)
- Kazuo Tamura
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan.
| | - Motohide Kaneda
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Mashu Futagawa
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Miho Takeshita
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Sanghyuk Kim
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Mina Nakama
- Division of Clinical Genetics, Gifu University Hospital, Gifu, Japan
| | - Norihito Kawashita
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
| | - Junko Tatsumi-Miyajima
- Division of Medical Genetics, Master of Science, Graduate School of Science and Engineering Research, Kindai University, Higashiosaka, Japan
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Human Exonuclease 1 (EXO1) Regulatory Functions in DNA Replication with Putative Roles in Cancer. Int J Mol Sci 2018; 20:ijms20010074. [PMID: 30585186 PMCID: PMC6337416 DOI: 10.3390/ijms20010074] [Citation(s) in RCA: 39] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2018] [Revised: 12/12/2018] [Accepted: 12/19/2018] [Indexed: 12/11/2022] Open
Abstract
Human exonuclease 1 (EXO1), a 5'→3' exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.
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7
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Liu J, Lee JB, Fishel R. Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair. J Mol Biol 2018; 430:4456-4468. [PMID: 29864444 PMCID: PMC6461355 DOI: 10.1016/j.jmb.2018.05.039] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 05/09/2018] [Accepted: 05/28/2018] [Indexed: 02/06/2023]
Abstract
DNA mismatch repair (MMR) is a DNA excision-resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.
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Affiliation(s)
- Jiaquan Liu
- Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, 43210, OH, USA
| | - Jong-Bong Lee
- Department of Physics, Pohang University of Science and Technology (POSTECH), 790-784, Pohang, Korea; Interdisciplinary Bioscience and Bioengineering, POSTECH, 790-784, Pohang, Korea.
| | - Richard Fishel
- Department of Cancer Biology and Genetics, The Ohio State University Wexner Medical Center, Columbus, 43210, OH, USA.
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Dai Y, Tang Z, Yang Z, Zhang L, Deng Q, Zhang X, Yu Y, Liu X, Zhu J. EXO1 overexpression is associated with poor prognosis of hepatocellular carcinoma patients. Cell Cycle 2018; 17:2386-2397. [PMID: 30328366 PMCID: PMC6237436 DOI: 10.1080/15384101.2018.1534511] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2018] [Revised: 09/17/2018] [Accepted: 10/05/2018] [Indexed: 02/08/2023] Open
Abstract
The roles of exonuclease 1 (EXO1) in hepatocellular carcinoma (HCC) tumorigenesis and progression remain unclear. This study aimed to assess the prognostic value and therapeutic potential of EXO1 in HCC. Exo1 gene copy numbers were obtained from three Oncomine microarray datasets (n = 447). EXO1 mRNA expression was validated by semi-quantitative PCR and QuantiGene® 2.0 assays. Cell growth curve and colony formation were performed to asses the cell proliferation. Clonogenic assay, flow cytometry, and immunofluorescence were adopted to acess the effects of EXO1 knockdown and radiation on cell survival, cell cycle distribution and DNA repair. Western blots were performed to reveal the related mechanism. A significant copy number variation (CNV) of the Exo1 gene was found in HCC specimens in three separate sets of published microarray data. In the 143 cases treated by our team, EXO1 expression levels were elevated (86.71%, 124/143). In addition, EXO1 overexpression was correlated with larger tumor size (P = 0.002), increased lymph node metastasis (P=0.033) and lower Edmondson grade (P = 0.018). High EXO1 expression unfavorably affected overall survival (OS) (P = 0.009). Both univariate and multivariate Cox regression analyses identified EXO1 as an independent predictor of OS (univariate, P = 0.012; multivariate, P = 0.039). Silencing of EXO1 in vitro reduced cell proliferation. EXO1 knockdown further suppressed clonogenic cell survival, abrogated radiation-induced G2/M phase arrest, and enhanced γ-H2AX foci after exposure to irradiation. The accumulation of ataxiatelangiectasia mutated (ATM) might partially regulate the EXO1 related radiosensitivity. In summary, EXO1 could be a promising prognostic marker, with a potential therapeutic value in HCC.
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Affiliation(s)
- Yaoyao Dai
- Department of Hepatology, Shanghai municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Zuxiong Tang
- Department of General surgery, the first affiliated hospital of soochow university, suzhou, China
| | - Zongguo Yang
- Department of Central Laboratory Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Lan Zhang
- Department of Hepatology, Shanghai municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Qing Deng
- Department of Central Laboratory Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Xiaofeng Zhang
- Department of Central Laboratory Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Yongchun Yu
- Department of Central Laboratory Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Xing Liu
- Department of Central Laboratory Medicine, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
- Shanghai Biochip Corporation LTD./National Engineering Center for Biochip at Shanghai, Shanghai, China
| | - Junfeng Zhu
- Department of Hepatology, Shanghai municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
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Lamba A, Parekh P, Dvorak CT, Karlitz JJ. Pedigree analysis supports a correlation between an AXIN2 variant and polyposis/colorectal cancer. World J Med Genet 2018; 8:1-4. [DOI: 10.5496/wjmg.v8.i1.1] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2017] [Revised: 11/18/2017] [Accepted: 12/29/2017] [Indexed: 02/06/2023] Open
Abstract
We present a patient with a history of colonic polyposis and family history significant for colon polyps and colorectal cancer (CRC). The patient and the family also had a history of bone loss of the jaw and early tooth loss, consistent with oligodontia. Genetic testing revealed the patient to have a previously unpublished variant of unknown significance (VUS) in the AXIN2 gene. These clinical findings have been demonstrated previously in only two other families, both of which exhibited oligodontia, colorectal neoplasia (polyps and cancer) and a heterozygous mutation in AXIN2. The AXIN2 protein is component of the Wnt pathway, which is known to be vital for organism development and cellular homeostasis. Alterations of the Wnt pathway lead to cell proliferation and neoplasm, in addition to agenesis of physical structures (such as teeth). The analysis of our pedigree further supports an association between colonic neoplasm (polyposis and CRC), the AXIN2 gene in general, and this particular VUS. It also highlights the importance of analyzing and disseminating information on pedigrees with less commonly encountered genomic abnormalities so that genotypic-phenotypic correlations can be solidified.
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Affiliation(s)
- Amrit Lamba
- Department of Internal Medicine, Tulane University, New Orleans, LA 70112, United States
| | - Parth Parekh
- Department of Gastroenterology, Carillion Clinic, Roanoke, VA 24016, United States
| | - Chris T Dvorak
- Department of Genetics, Tulane University, New Orleans, LA 70112, United States
| | - Jordan J Karlitz
- Department of Gastroenterology, Tulane University, New Orleans, LA 70112, United States
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Tan S, Qin R, Zhu X, Tan C, Song J, Qin L, Liu L, Huang X, Li A, Qiu X. Associations between single-nucleotide polymorphisms of human exonuclease 1 and the risk of hepatocellular carcinoma. Oncotarget 2018; 7:87180-87193. [PMID: 27894089 PMCID: PMC5349980 DOI: 10.18632/oncotarget.13517] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Accepted: 10/17/2016] [Indexed: 01/27/2023] Open
Abstract
Human exonuclease 1 (hEXO1) is an important nuclease involved in mismatch repair system that contributes to maintain genomic stability and modulate DNA recombination. This study is aimed to explore the associations between single-nucleotide polymorphisms (SNPs) of hEXO1 and the hereditary susceptibility of hepatocellular carcinoma (HCC). SNPs rs1047840, rs1776148, rs3754093, rs4149867, rs4149963, and rs1776181 of hEXO1 were examined from a hospital-based case-control study including 1,196 cases (HCC patients) and 1,199 controls (non-HCC patients) in Guangxi, China. We found the rs3754093 AG genotype decreased the risk of HCC (OR=0.714, 95% CI: 0.539∼0.946). According to the results of stratification analysis, rs3754093 mutant genotype AG/GG decreased the risk of HCC with some HCC protective factors such as non-smoking, non-alcohol consumption and non-HCC family history, but also decreased the risk of HCC with HBV infection. Moreover, it was correlated to non-tumor metastasis and increased the survival of HCC patients. The results from gene-environment interaction assay indicated all hEXO1 SNPs interacted with smoking, alcohol consumption, HBV infection in pathogenesis of HCC. However, gene-gene interaction assay suggested the interaction between rs3754093 and other 5 SNPs were associated with reducing the HCC risk. These results suggest rs3754093 exhibits a protective activity to decrease the incidence risk of HCC in Guangxi, China. In addition, all SNPs in this study interacted with environment risk factors in pathogenesis of HCC.
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Affiliation(s)
- Shengkui Tan
- Department of Epidemiology, School of Public Health, Guilin Medical University, Guilin 541004, Guangxi, People's Republic of China
| | - Ruoyun Qin
- Department of Epidemiology, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, People's Republic of China
| | - Xiaonian Zhu
- Department of Epidemiology, School of Public Health, Guilin Medical University, Guilin 541004, Guangxi, People's Republic of China
| | - Chao Tan
- Guangxi Center for Disease Prevention and Control, Nanning 530021, Guangxi, People's Republic of China
| | - Jiale Song
- Department of Epidemiology, School of Public Health, Guilin Medical University, Guilin 541004, Guangxi, People's Republic of China
| | - Linyuan Qin
- Department of Epidemiology, School of Public Health, Guilin Medical University, Guilin 541004, Guangxi, People's Republic of China
| | - Liu Liu
- Department of Epidemiology, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, People's Republic of China
| | - Xiong Huang
- Department of Epidemiology, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, People's Republic of China
| | - Anhua Li
- Guangxi Center for Disease Prevention and Control, Nanning 530021, Guangxi, People's Republic of China
| | - Xiaoqiang Qiu
- Department of Epidemiology, School of Public Health, Guangxi Medical University, Nanning 530021, Guangxi, People's Republic of China
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Keijzers G, Liu D, Rasmussen LJ. Exonuclease 1 and its versatile roles in DNA repair. Crit Rev Biochem Mol Biol 2016; 51:440-451. [PMID: 27494243 DOI: 10.1080/10409238.2016.1215407] [Citation(s) in RCA: 38] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification.
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Affiliation(s)
- Guido Keijzers
- a Department of Cellular and Molecular Medicine , Center for Healthy Aging, University of Copenhagen , Copenhagen , Denmark
| | - Dekang Liu
- a Department of Cellular and Molecular Medicine , Center for Healthy Aging, University of Copenhagen , Copenhagen , Denmark
| | - Lene Juel Rasmussen
- a Department of Cellular and Molecular Medicine , Center for Healthy Aging, University of Copenhagen , Copenhagen , Denmark
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12
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Chen ZY, Zheng SR, Zhong JH, Zhuang XD, Zhou JY. Association between three exonuclease 1 polymorphisms and cancer risks: a meta-analysis. Onco Targets Ther 2016; 9:899-910. [PMID: 26966378 PMCID: PMC4770079 DOI: 10.2147/ott.s95258] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
To date, the results of studies exploring the relation between exonuclease 1 (Exo1) polymorphisms and cancer risks have differed. In this study, we performed a meta-analysis to investigate the effect of the three most extensively studied Exo1 polymorphisms (Pro757Leu, Glu589Lys, and Glu670Gly) on cancer susceptibility. The related studies published before August 5, 2015, were collected by searching the PubMed and EMBASE databases. We found 16 publications containing studies that were eligible for our study, including 10 studies for Pro757Leu polymorphism (4,093 cases and 3,834 controls), 12 studies for Glu589Lys polymorphism (6,479 cases and 6,550 controls), and 7 studies for Glu670Gly polymorphism (3,700 cases and 3,496 controls). Pooled odds ratios and 95% confidence intervals were used to assess the strength of the associations, and all the statistical analyses were calculated using the software program STATA version 12.0. Our results revealed that the Pro757Leu polymorphism was significantly associated with a reduced cancer risk, whereas an inverse association was found for the Glu589Lys polymorphism. Furthermore, subgroup analysis of smoking status indicated that the Glu589Lys polymorphism was significantly associated with an increased cancer risk in smokers, but not in nonsmokers. However, no evidence was found for an association between the Glu670Gly polymorphism and cancer risk. In conclusion, this meta-analysis suggests that the Pro757Leu polymorphism may provide protective effects against cancer, while the Glu589Lys polymorphism may be a risk factor for cancer. Moreover, the Glu670Gly polymorphism may have no influence on cancer susceptibility. In the future, large-scaled and well-designed studies are needed to achieve a more precise and comprehensive result.
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Affiliation(s)
- Zi-Yu Chen
- Department of Clinical Medicine, The First Clinical Medical College, Southern Medical University, Guangzhou, People's Republic of China; Institute of Genetic Engineering, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People's Republic of China
| | - Si-Rong Zheng
- Institute of Genetic Engineering, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People's Republic of China
| | - Jie-Hui Zhong
- Department of Clinical Medicine, The First Clinical Medical College, Southern Medical University, Guangzhou, People's Republic of China; Institute of Genetic Engineering, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People's Republic of China
| | - Xiao-Duan Zhuang
- Department of Clinical Medicine, The First Clinical Medical College, Southern Medical University, Guangzhou, People's Republic of China; Institute of Genetic Engineering, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People's Republic of China
| | - Jue-Yu Zhou
- Institute of Genetic Engineering, School of Basic Medical Sciences, Southern Medical University, Guangzhou, People's Republic of China
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13
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Schmidt TT, Hombauer H. Visualization of mismatch repair complexes using fluorescence microscopy. DNA Repair (Amst) 2016; 38:58-67. [DOI: 10.1016/j.dnarep.2015.11.014] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2015] [Revised: 09/30/2015] [Accepted: 11/30/2015] [Indexed: 11/15/2022]
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14
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Talseth-Palmer BA, Bauer DC, Sjursen W, Evans TJ, McPhillips M, Proietto A, Otton G, Spigelman AD, Scott RJ. Targeted next-generation sequencing of 22 mismatch repair genes identifies Lynch syndrome families. Cancer Med 2016; 5:929-41. [PMID: 26811195 PMCID: PMC4864822 DOI: 10.1002/cam4.628] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Revised: 11/09/2015] [Accepted: 11/30/2015] [Indexed: 01/04/2023] Open
Abstract
Causative germline mutations in mismatch repair (MMR) genes can only be identified in ~50% of families with a clinical diagnosis of the inherited colorectal cancer (CRC) syndrome hereditary nonpolyposis colorectal cancer (HNPCC)/Lynch syndrome (LS). Identification of these patients are critical as they are at substantially increased risk of developing multiple primary tumors, mainly colorectal and endometrial cancer (EC), occurring at a young age. This demonstrates the need to develop new and/or more thorough mutation detection approaches. Next‐generation sequencing (NGS) was used to screen 22 genes involved in the DNA MMR pathway in constitutional DNA from 14 HNPCC and 12 sporadic EC patients, plus 2 positive controls. Several softwares were used for analysis and functional annotation. We identified 5 exonic indel variants, 42 exonic nonsynonymous single‐nucleotide variants (SNVs) and 1 intronic variant of significance. Three of these variants were class 5 (pathogenic) or class 4 (likely pathogenic), 5 were class 3 (uncertain clinical relevance) and 40 were classified as variants of unknown clinical significance. In conclusion, we have identified two LS families from the sporadic EC patients, one without a family history of cancer, supporting the notion for universal MMR screening of EC patients. In addition, we have detected three novel class 3 variants in EC cases. We have, in addition discovered a polygenic interaction which is the most likely cause of cancer development in a HNPCC patient that could explain previous inconsistent results reported on an intronic EXO1 variant.
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Affiliation(s)
- Bente A Talseth-Palmer
- School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, New South Wales, Australia.,Centre for Information-Based Medicine, Hunter Medical Research Institute, Newcastle, New South Wales, Australia
| | - Denis C Bauer
- CSIRO Digital Productivity, Sydney, New South Wales, Australia
| | - Wenche Sjursen
- Department of Laboratory Medicine Children's and Women's Health, Norwegian University of Science and Technology, Trondheim, Norway.,Department of Pathology and Medical Genetics, St Olavs University Hospital, Trondheim, Norway
| | - Tiffany J Evans
- School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, New South Wales, Australia.,Centre for Information-Based Medicine, Hunter Medical Research Institute, Newcastle, New South Wales, Australia
| | - Mary McPhillips
- Hunter Area Pathology Service, Pathology North, Hunter New England Area Health, Newcastle, New South Wales, Australia
| | - Anthony Proietto
- Hunter Centre for Gynaecological Cancer, Hunter New England Area Health, Newcastle, New South Wales, Australia.,School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, New South Wales, Australia
| | - Geoffrey Otton
- Hunter Centre for Gynaecological Cancer, Hunter New England Area Health, Newcastle, New South Wales, Australia.,School of Medicine and Public Health, Faculty of Health and Medicine, University of Newcastle, New South Wales, Australia
| | - Allan D Spigelman
- Hunter Family Cancer Service, Hunter New England Area Health, Newcastle, New South Wales, Australia.,St Vincent's Hospital Clinical School, University of NSW and Hospital Cancer Genetics Clinic, The Kinghorn Cancer Centre, Sydney, New South Wales, Australia
| | - Rodney J Scott
- School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, Newcastle, New South Wales, Australia.,Centre for Information-Based Medicine, Hunter Medical Research Institute, Newcastle, New South Wales, Australia.,Hunter Area Pathology Service, Pathology North, Hunter New England Area Health, Newcastle, New South Wales, Australia
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15
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Heinen CD. Mismatch repair defects and Lynch syndrome: The role of the basic scientist in the battle against cancer. DNA Repair (Amst) 2015; 38:127-134. [PMID: 26710976 DOI: 10.1016/j.dnarep.2015.11.025] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2015] [Revised: 08/21/2015] [Accepted: 11/30/2015] [Indexed: 12/24/2022]
Abstract
We have currently entered a genomic era of cancer research which may soon lead to a genomic era of cancer treatment. Patient DNA sequencing information may lead to a personalized approach to managing an individual's cancer as well as future cancer risk. The success of this approach, however, begins not necessarily in the clinician's office, but rather at the laboratory bench of the basic scientist. The basic scientist plays a critical role since the DNA sequencing information is of limited use unless one knows the function of the gene that is altered and the manner by which a sequence alteration affects that function. The role of basic science research in aiding the clinical management of a disease is perhaps best exemplified by considering the case of Lynch syndrome, a hereditary disease that predisposes patients to colorectal and other cancers. This review will examine how the diagnosis, treatment and even prevention of Lynch syndrome-associated cancers has benefitted from extensive basic science research on the DNA mismatch repair genes whose alteration underlies this condition.
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Affiliation(s)
- Christopher D Heinen
- Center for Molecular Medicine and Neag Comprehensive Cancer Center, University of Connecticut Health, Farmington, CT 06030, USA.
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16
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Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2015; 2016:3125989. [PMID: 26649135 PMCID: PMC4663340 DOI: 10.1155/2016/3125989] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/21/2015] [Revised: 07/08/2015] [Accepted: 07/27/2015] [Indexed: 12/12/2022]
Abstract
DNA oxidative lesions are widely considered as a potential risk factor for colorectal cancer development. The aim of this work was to determine the role of the efficiency of base excision repair, both in lymphocytes and in epithelial tissue, in patients with CRC and healthy subjects. SNPs were identified within genes responsible for steps following glycosylase action in BER, and patients and healthy subjects were genotyped. A radioisotopic BER assay was used for assessing repair efficiency and TaqMan for genotyping. Decreased BER activity was observed in lymphocyte extract from CRC patients and in cancer tissue extract, compared to healthy subjects. In addition, polymorphisms of EXO1, LIG3, and PolB may modulate the risk of colorectal cancer by decreasing (PolB) or increasing (LIG3 and EXO1) the chance of malignant transformation.
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17
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Ghafouri-Fard S, Fardaei M, Lankarani KB, Miryounesi M. Segregation of a novel MLH1 mutation in an Iranian Lynch syndrome family. Gene 2015; 570:304-305. [PMID: 26149658 DOI: 10.1016/j.gene.2015.07.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Revised: 06/30/2015] [Accepted: 07/01/2015] [Indexed: 02/08/2023]
Abstract
Lynch syndrome is an autosomal dominant disorder caused by germline mutations in mismatch repair (MMR) genes. The main feature of this disorder is an early onset of hereditary colorectal cancer in addition to other cancers arising from different tissues. Here, we report an Iranian family with several members affected with Lynch syndrome related cancers. Exome sequencing with focus on 14 genes related with hereditary colorectal cancer has shown a novel mutation in exon 19 of MLH1 gene (c.2133delC, p.Trp712Gly fs*71). This mutation is located in a region coding for the functional domain for the interaction with MLH3/PMS1/PMS2. As some clinical aspects of the disorder have been shown to be associated with certain mutations, identification of causative mutation in each family has implications for surveillance protocols.
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Affiliation(s)
- Soudeh Ghafouri-Fard
- Department of Medical Genetics, Shahid Beheshti University of Medical sciences, Tehran, Iran
| | - Majid Fardaei
- Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, Iran
| | | | - Mohammad Miryounesi
- Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
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18
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Human exonuclease 1 (EXO1) activity characterization and its function on flap structures. Biosci Rep 2015; 35:BSR20150058. [PMID: 26182368 PMCID: PMC4613700 DOI: 10.1042/bsr20150058] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Accepted: 04/24/2015] [Indexed: 11/26/2022] Open
Abstract
We report biochemical characterization of human full-length EXO1 including thermodynamic stability and flap activity on DNA flap structures. Our results reveal novel mechanistic insights into the processing of flap structures and a possible role of EXO1 in strand displacement. Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5′ flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement.
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19
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Petersen SM, Dandanell M, Rasmussen LJ, Gerdes AM, Krogh LN, Bernstein I, Okkels H, Wikman F, Nielsen FC, Hansen TVO. Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients. BMC MEDICAL GENETICS 2013; 14:103. [PMID: 24090359 PMCID: PMC3850734 DOI: 10.1186/1471-2350-14-103] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/11/2013] [Accepted: 09/25/2013] [Indexed: 12/12/2022]
Abstract
Background Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Methods Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. Results We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T). Conclusions In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.
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Affiliation(s)
- Sanne M Petersen
- Center for Genomic Medicine, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
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20
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Different strategies to overcome multidrug resistance in cancer. Biotechnol Adv 2013; 31:1397-407. [PMID: 23800690 DOI: 10.1016/j.biotechadv.2013.06.004] [Citation(s) in RCA: 179] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2013] [Revised: 06/06/2013] [Accepted: 06/14/2013] [Indexed: 01/21/2023]
Abstract
The risk of acquisition of resistance to chemotherapy remains a major hurdle in the management of various types of cancer patients. Several cellular and noncellular mechanisms are involved in developing both intrinsic and acquired resistance in cancer cells toward chemotherapy. This review covers the various multidrug resistance (MDR) mechanisms observed in cancer cells as well as the various strategies developed to overcome these MDR mechanisms. Extensive studies have been conducted during the last several decades to enhance the efficacy of chemotherapy by suppressing or evading these MDR mechanisms including the use of new anticancer drugs that could escape from the efflux reaction, MDR modulators or chemosensitizers, multifunctional nanocarriers, and RNA interference (RNAi) therapy.
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21
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MSH6 mutations are frequent in hereditary nonpolyposis colorectal cancer families with normal pMSH6 expression as detected by immunohistochemistry. Appl Immunohistochem Mol Morphol 2013; 20:470-7. [PMID: 22495361 DOI: 10.1097/pai.0b013e318249739b] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
INTRODUCTION Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant condition accounting for 2% to 4% of all colorectal cancer cases worldwide. Families with germ line mutations in 1 of 6 mismatch repair genes are known as Lynch syndrome families. The largest number of mutations has been detected in the mismatch repair genes MLH1 and MSH2, but several mutations in MSH6 have also been demonstrated. AIM : Whether HNPCC families are screened for mutations in mismatch repair genes often relies on their immunohistochemical profile. The aim of the present study was to evaluate this approach in Lynch families carrying mutations in MSH6. MATERIALS AND METHODS Results of the screening of the MSH6 gene in HNPCC families were compared with those obtained on immunohistochemical protein analysis. RESULTS In 56 (7%) of 815 families, at least 1 MSH6 mutation, 23 definitively pathogenic mutations and 38 missense mutations or unclassified variants, and several polymorphisms in the MSH6 gene were detected. In families carrying a pathogenic MSH6 mutation, 69.6% of 23 colon adenocarcinomas showed absence of pMSH6 in tumor tissue by immunohistochemical analysis. In 34.5%, all proteins could be detected, whereas in 34.5% pMSH6 was present and pMLH1/pPMS2 was absent. CONCLUSIONS If genetic screening of HNPCC families depended on immunohistochemical results, a substantial number of families harboring a pathogenic mutation in MSH6 and the vast majority of families harboring an MSH6 unclassified variant would not be detected.
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22
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Panigrahi GB, Slean MM, Simard JP, Pearson CE. Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats. J Biol Chem 2012; 287:41844-50. [PMID: 23086927 DOI: 10.1074/jbc.m112.420398] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.
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Affiliation(s)
- Gagan B Panigrahi
- Department of Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
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23
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Exonuclease 1 (Exo1) is required for activating response to S(N)1 DNA methylating agents. DNA Repair (Amst) 2012; 11:951-64. [PMID: 23062884 DOI: 10.1016/j.dnarep.2012.09.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2012] [Revised: 09/08/2012] [Accepted: 09/11/2012] [Indexed: 11/23/2022]
Abstract
S(N)1 DNA methylating agents are genotoxic agents that methylate numerous nucleophilic centers within DNA including the O(6) position of guanine (O(6)meG). Methylation of this extracyclic oxygen forces mispairing with thymine during DNA replication. The mismatch repair (MMR) system recognizes these O(6)meG:T mispairs and is required to activate DNA damage response (DDR). Exonuclease I (EXO1) is a key component of MMR by resecting the damaged strand; however, whether EXO1 is required to activate MMR-dependent DDR remains unknown. Here we show that knockdown of the mouse ortholog (mExo1) in mouse embryonic fibroblasts (MEFs) results in decreased G2/M checkpoint response, limited effects on cell proliferation, and increased cell viability following exposure to the S(N)1 methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), establishing a phenotype paralleling MMR deficiency. MNNG treatment induced formation of γ-H2AX foci with which EXO1 co-localized in MEFs, but mExo1-depleted MEFs displayed a significant diminishment of γ-H2AX foci formation. mExo1 depletion also reduced MSH2 association with DNA duplexes containing G:T mismatches in vitro, decreased MSH2 association with alkylated chromatin in vivo, and abrogated MNNG-induced MSH2/CHK1 interaction. To determine if nuclease activity is required to activate DDR we stably overexpressed a nuclease defective form of human EXO1 (hEXO1) in mExo1-depleted MEFs. These experiments indicated that expression of wildtype and catalytically null hEXO1 was able to restore normal response to MNNG. This study indicates that EXO1 is required to activate MMR-dependent DDR in response to S(N)1 methylating agents; however, this function of EXO1 is independent of its nucleolytic activity.
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Andersen SD, Liberti SE, Lützen A, Drost M, Bernstein I, Nilbert M, Dominguez M, Nyström M, Hansen TVO, Christoffersen JW, Jäger AC, de Wind N, Nielsen FC, Tørring PM, Rasmussen LJ. Functional characterization ofMLH1missense variants identified in lynch syndrome patients. Hum Mutat 2012; 33:1647-55. [DOI: 10.1002/humu.22153] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2011] [Accepted: 06/03/2012] [Indexed: 01/30/2023]
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25
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Heinen CD, Juel Rasmussen L. Determining the functional significance of mismatch repair gene missense variants using biochemical and cellular assays. Hered Cancer Clin Pract 2012; 10:9. [PMID: 22824075 PMCID: PMC3434035 DOI: 10.1186/1897-4287-10-9] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2012] [Accepted: 06/28/2012] [Indexed: 12/15/2022] Open
Abstract
With the discovery that the hereditary cancer susceptibility disease Lynch syndrome (LS) is caused by deleterious germline mutations in the DNA mismatch repair (MMR) genes nearly 20 years ago, genetic testing can now be used to diagnose this disorder in patients. A definitive diagnosis of LS can direct how clinicians manage the disease as well as prevent future cancers for the patient and their families. A challenge emerges, however, when a germline missense variant is identified in a MMR gene in a suspected LS patient. The significance of a single amino acid change in these large repair proteins is not immediately obvious resulting in them being designated variants of uncertain significance (VUS). One important strategy for resolving this uncertainty is to determine whether the variant results in a non-functional protein. The ability to reconstitute the MMR reaction in vitro has provided an important experimental tool for studying the functional consequences of VUS. However, beyond this repair assay, a number of other experimental methods have been developed that allow us to test the effect of a VUS on discrete biochemical steps or other aspects of MMR function. Here, we describe some of these assays along with the challenges of using such assays to determine the functional consequences of MMR VUS which, in turn, can provide valuable insight into their clinical significance. With increased gene sequencing in patients, the number of identified VUS has expanded dramatically exacerbating this problem for clinicians. However, basic science research laboratories around the world continue to expand our knowledge of the overall MMR molecular mechanism providing new opportunities to understand the functional significance, and therefore pathogenic significance, of VUS.
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Affiliation(s)
- Christopher D Heinen
- Neag Comprehensive Cancer Center and Center for Molecular Medicine, University of Connecticut Health Center, 233 Farmington Avenue, ML3101 Farmington, CT, USA.
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26
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Ali H, Olatubosun A, Vihinen M. Classification of mismatch repair gene missense variants with PON-MMR. Hum Mutat 2012; 33:642-50. [PMID: 22290698 DOI: 10.1002/humu.22038] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Numerous mismatch repair (MMR) gene variants have been identified in Lynch syndrome and other cancer patients, but knowledge about their pathogenicity is frequently missing. The diagnosis and treatment of patients would benefit from knowing which variants are disease related. Bioinformatic approaches are well suited to the problem and can handle large numbers of cases. Functional effects were revealed based on literature for 168 MMR missense variants. Performance of numerous prediction methods was tested with this dataset. Among the tested tools, only the results of tolerance prediction methods correlated to functional information, however, with poor performance. Therefore, a novel consensus-based predictor was developed. The novel prediction method, pathogenic-or-not mismatch repair (PON-MMR), achieved accuracy of 0.87 and Matthews correlation coefficient of 0.77 on the experimentally verified variants. When applied to 616 MMR cases with unknown effects, 81 missense variants were predicted to be pathogenic and 167 neutral. With PON-MMR, the number of MMR missense variants with unknown effect was reduced by classifying a large number of cases as likely pathogenic or benign. The results can be used, for example, to prioritize cases for experimental studies and assist in the classification of cases.
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Affiliation(s)
- Heidi Ali
- Institute of Biomedical Technology, FI-33014 University of Tampere, Finland
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27
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Brieger A, Plotz G, Hinrichsen I, Passmann S, Adam R, Zeuzem S. C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins. PLoS One 2012; 7:e31863. [PMID: 22348133 PMCID: PMC3279419 DOI: 10.1371/journal.pone.0031863] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2011] [Accepted: 01/13/2012] [Indexed: 12/22/2022] Open
Abstract
The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
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Affiliation(s)
- Angela Brieger
- Department of Medicine I, University of Frankfurt/M., Frankfurt, Germany.
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28
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The functions of MutL in mismatch repair: the power of multitasking. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2012; 110:41-70. [PMID: 22749142 DOI: 10.1016/b978-0-12-387665-2.00003-1] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
DNA mismatch repair enhances genomic stability by correcting errors that have escaped polymerase proofreading. One of the critical steps in DNA mismatch repair is discriminating the new from the parental DNA strand as only the former needs repair. In Escherichia coli, the latent endonuclease MutH carries out this function. However, most prokaryotes and all eukaryotes lack a mutH gene. MutL is a key component of this system that mediates protein-protein interactions during mismatch recognition, strand discrimination, and strand removal. Hence, it had long been thought that the primary function of MutL was coordinating sequential mismatch repair steps. However, recent studies have revealed that most MutL homologs from organisms lacking MutH encode a conserved metal-binding motif associated with a weak endonuclease activity. As MutL homologs bearing this activity are found only in organisms relying on MutH-independent DNA mismatch repair, this finding unveils yet another crucial function of the MutL protein at the strand discrimination step. In this chapter, we review recent functional and structural work aimed at characterizing the multiple functions of MutL and discuss how the endonuclease activity of MutL is regulated by other repair factors.
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Bayram S, Akkız H, Bekar A, Akgöllü E, Yıldırım S. The significance of Exonuclease 1 K589E polymorphism on hepatocellular carcinoma susceptibility in the Turkish population: a case-control study. Mol Biol Rep 2011; 39:5943-51. [PMID: 22205538 DOI: 10.1007/s11033-011-1406-x] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2011] [Accepted: 12/17/2011] [Indexed: 02/07/2023]
Abstract
Exonuclease 1 (Exo 1) is an important nuclease involved in mismatch repair system that contributes to maintain genomic stability, to modulate DNA recombination, and to mediate cell cycle arrest. A guanine (G)/adenine (A) common single nucleotide polymorphism at first position of codon 589 in Exo 1 gene determines a glutamic acid (Glu, E) to lysine (Lys, K) (K589E) aminoacidic substitution which may alter cancer risk by influencing the activity of Exo 1 protein. Exo 1 K589E polymorphism has been studied in various cancers, but its association with hepatocellular carcinoma (HCC) has yet to be investigated. To determine the association of the Exo 1 K589E polymorphism with the risk of HCC development in a Turkish population, a hospital-based case-control study was designed consisting of 224 subjects with HCC and 224 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the Exo 1 K589E polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay. Our data shows that the Lys/Lys genotype of the Exo 1 K589E polymorphism is associated with increased risk of HCC development in this Turkish population [odds ratio (OR) = 2.15, 95% confidence interval (CI): 1.13-4.09, P = 0.02]. Furthermore, according to stratified analysis, a significant association was observed between the homozygote Lys/Lys genotype and HCC risk in the subgroups of male gender (OR = 2.67, 95% CI: 1.27-5.61, P = 0.009) and patients with non-viral-related HCC (OR = 3.14, 95% CI: 1.09-8.99, P = 0.03). Because our results suggest for the first time that the Lys/Lys homozygote genotype of Exo 1 K589E polymorphism may be a genetic susceptibility factor for HCC in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.
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Affiliation(s)
- Süleyman Bayram
- Department of Nursing, Adıyaman School of Health, Adıyaman University, 02040, Adıyaman, Turkey.
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Liberti SE, Andersen SD, Wang J, May A, Miron S, Perderiset M, Keijzers G, Nielsen FC, Charbonnier JB, Bohr VA, Rasmussen LJ. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks. DNA Repair (Amst) 2010; 10:73-86. [PMID: 20970388 DOI: 10.1016/j.dnarep.2010.09.023] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2010] [Revised: 09/26/2010] [Accepted: 09/28/2010] [Indexed: 01/23/2023]
Abstract
Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
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Affiliation(s)
- Sascha E Liberti
- Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark
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31
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Leong V, Lorenowicz J, Kozij N, Guarné A. Nuclear import of human MLH1, PMS2, and MutLalpha: redundancy is the key. Mol Carcinog 2009; 48:742-50. [PMID: 19148896 DOI: 10.1002/mc.20514] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
DNA mismatch repair maintains genomic stability by correcting errors that have escaped polymerase proofreading. Defects on mismatch repair genes lead to an increased mutation rate, microsatellite instability and predisposition to human non-polyposis colorectal cancer (HNPCC). Human MutLalpha is a heterodimer formed by the interaction of MLH1 and PMS2 that coordinates a series of key events in mismatch repair. It has been proposed that nuclear import of MutLalpha may be the first regulatory step on the activation of the mismatch repair pathway. Using confocal microscopy and mismatch repair deficient cells, we have identified the sequence determinants that drive nuclear import of human MLH1, PMS2, and MutLalpha. Transient transfection of the individual proteins reveals that MLH1 has a bipartite and PMS2 has a single monopartite nuclear localization signal. Although dimerization is not required for nuclear localization, the MutLalpha heterodimer is imported more efficiently than the MLH1 or PMS2 monomers. Interestingly, the bipartite localization signal of MLH1 can direct import of MutLalpha even when PMS2 encompasses a mutated localization signal. Hence we conclude that the presence of redundant nuclear localization signals guarantees nuclear transport of MutLalpha and, consequently, efficient mismatch repair.
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Affiliation(s)
- Vivian Leong
- Department of Biochemistry and Biomedical Sciences, McMaster University, Ontario, Canada
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32
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Tsai MH, Tseng HC, Liu CS, Chang CL, Tsai CW, Tsou YA, Wang RF, Lin CC, Wang HC, Chiu CF, Bau DT. Interaction of Exo1 genotypes and smoking habit in oral cancer in Taiwan. Oral Oncol 2009; 45:e90-4. [PMID: 19515603 DOI: 10.1016/j.oraloncology.2009.03.011] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2009] [Revised: 03/19/2009] [Accepted: 03/23/2009] [Indexed: 12/15/2022]
Abstract
Exonuclease 1 (Exo1) is an important nuclease involved in the mismatch repair system that helps to maintain genomic stability, to modulate DNA recombination, and to mediate cell cycle arrest. Potential polymorphisms in Exo1 may alter cancer risks by influencing the repair activity of Exo1. Therefore, we hypothesized that single-nucleotide polymorphisms in Exo1 were associated with the risk of oral cancer. In this hospital-based study, the associations of Exo1 A-1419G (rs3754093), C-908G (rs10802996), A238G (rs1776177), C498T (rs1635517), K589E (rs1047840), G670E (rs1776148), C723R (rs1635498), L757P (rs9350) and C3114T (rs851797) polymorphisms with oral cancer risk in a central Taiwan population were investigated. In total, 680 patients with oral cancer and 680 age- and gender-matched healthy controls recruited from the China Medical University Hospital were genotyped. A significantly different distribution is found in the frequency of the Exo1 K589E genotype, but not the other genotypes, between the oral cancer and control groups. The A allele Exo1 K589E conferred a significant (P=6.18E-8) increased risk of oral cancer. Gene-environment interactions with smoking were significant for Exo1 K589E polymorphism (OR=2.509, 95% CI=1.914-3.287). Our results provide evidence that the A allele of the Exo1 K589E may be associated with the development of oral cancer.
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Affiliation(s)
- Ming-Hsui Tsai
- China Medical University Hospital, Taichung, Taiwan, ROC
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Knudsen NØ, Andersen SD, Lützen A, Nielsen FC, Rasmussen LJ. Nuclear translocation contributes to regulation of DNA excision repair activities. DNA Repair (Amst) 2009; 8:682-9. [PMID: 19376751 DOI: 10.1016/j.dnarep.2009.03.005] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2009] [Revised: 03/06/2009] [Accepted: 03/14/2009] [Indexed: 11/26/2022]
Abstract
DNA mutations are circumvented by dedicated specialized excision repair systems, such as the base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) pathways. Although the individual repair pathways have distinct roles in suppressing changes in the nuclear DNA, it is evident that proteins from the different DNA repair pathways interact [Y. Wang, D. Cortez, P. Yazdi, N. Neff, S.J. Elledge, J. Qin, BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures, Genes Dev. 14 (2000) 927-939; M. Christmann, M.T. Tomicic, W.P. Roos, B. Kaina, Mechanisms of human DNA repair: an update, Toxicology 193 (2003) 3-34; N.B. Larsen, M. Rasmussen, L.J. Rasmussen, Nuclear and mitochondrial DNA repair: similar pathways? Mitochondrion 5 (2005) 89-108]. Protein interactions are not only important for function, but also for regulation of nuclear import that is necessary for proper localization of the repair proteins. This review summarizes the current knowledge on nuclear import mechanisms of DNA excision repair proteins and provides a model that categorizes the import by different mechanisms, including classical nuclear import, co-import of proteins, and alternative transport pathways. Most excision repair proteins appear to contain classical NLS sequences directing their nuclear import, however, additional import mechanisms add alternative regulatory levels to protein import, indirectly affecting protein function. Protein co-import appears to be a mechanism employed by the composite repair systems NER and MMR to enhance and regulate nuclear accumulation of repair proteins thereby ensuring faithful DNA repair.
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Affiliation(s)
- Nina Østergaard Knudsen
- Department of Science, Systems and Models, Roskilde University, Universitetsvej 1, 4000 Roskilde, Denmark
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Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair. Mol Cell Biol 2008; 29:907-18. [PMID: 19015241 DOI: 10.1128/mcb.00945-08] [Citation(s) in RCA: 51] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.
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Couch FJ, Rasmussen LJ, Hofstra R, Monteiro ANA, Greenblatt MS, de Wind N. Assessment of functional effects of unclassified genetic variants. Hum Mutat 2008; 29:1314-26. [PMID: 18951449 PMCID: PMC2771414 DOI: 10.1002/humu.20899] [Citation(s) in RCA: 85] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Inherited predisposition to disease is often linked to reduced activity of a disease associated gene product. Thus, quantitation of the influence of inherited variants on gene function can potentially be used to predict the disease relevance of these variants. While many disease genes have been extensively characterized at the functional level, few assays based on functional properties of the encoded proteins have been established for the purpose of predicting the contribution of rare inherited variants to disease. Much of the difficulty in establishing predictive functional assays stems from the technical complexity of the assays. However, perhaps the most challenging aspect of functional assay development for clinical testing purposes is the absolute requirement for validation of the sensitivity and specificity of the assays and the determination of positive predictive values (PPVs) and negative predictive values (NPVs) of the assays relative to a "gold standard" measure of disease predisposition. In this commentary, we provide examples of some of the functional assays under development for several cancer predisposition genes (BRCA1, BRCA2, CDKN2A, and mismatch repair [MMR] genes MLH1, MSH2, MSH6, and PMS2) and present a detailed review of the issues associated with functional assay development. We conclude that validation is paramount for all assays that will be used for clinical interpretation of inherited variants of any gene, but note that in certain circumstances information derived from incompletely validated assays may be valuable for classification of variants for clinical purposes when used to supplement data derived from other sources.
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Affiliation(s)
- Fergus J Couch
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, USA.
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36
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Lützen A, de Wind N, Georgijevic D, Nielsen FC, Rasmussen LJ. Functional analysis of HNPCC-related missense mutations in MSH2. Mutat Res 2008; 645:44-55. [PMID: 18822302 DOI: 10.1016/j.mrfmmm.2008.08.015] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2008] [Revised: 07/25/2008] [Accepted: 08/26/2008] [Indexed: 12/28/2022]
Abstract
Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.
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Affiliation(s)
- Anne Lützen
- Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark
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37
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Nilbert M, Wikman FP, Hansen TVO, Krarup HB, Orntoft TF, Nielsen FC, Sunde L, Gerdes AM, Cruger D, Timshel S, Bisgaard ML, Bernstein I, Okkels H. Major contribution from recurrent alterations and MSH6 mutations in the Danish Lynch syndrome population. Fam Cancer 2008; 8:75-83. [PMID: 18566915 DOI: 10.1007/s10689-008-9199-3] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2008] [Accepted: 06/02/2008] [Indexed: 12/27/2022]
Abstract
An increasing number of mismatch-repair (MMR) gene mutations have been identified in hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome. This study presents the population-based Danish MMR gene mutation profile, which contains 138 different MMR gene alterations. Among these, 88 mutations in 164 families are considered pathogenic and an additional 50 variants from 76 families are considered to represent variants of unknown pathogenicity. The different MMR genes contribute to 40% (MSH2), 29% (MLH1), and 22% (MSH6) of the mutations and the Danish population thus shows a considerably higher frequency of MSH6 mutations than previously described. Although 69/88 (78%) pathogenic mutations were present in a single family, previously recognized recurrent/founder mutations were causative in 75/137 (55%) MLH1/MSH2 mutant families. In addition, the Danish MLH1 founder mutation c.1667+2_1667_+8TAAATCAdelinsATTT was identified in 14/58 (24%) MLH1 mutant families. The Danish Lynch syndrome population thus demonstrates that MSH6 mutations and recurrent/founder mutations have a larger contribution than previously recognized, which implies that the MSH6 gene should be included in routine diagnostics and suggests that directed analysis of recurrent/founder mutations may be feasible e.g. in families were diagnostic material is restricted to archival tissue.
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Affiliation(s)
- Mef Nilbert
- Clinical Research Centre and HNPCC-Register, Copenhagen University, Hvidovre University Hospital, Kettegård Allé 30, Hvidovre, 2650, Denmark.
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Sacho EJ, Kadyrov FA, Modrich P, Kunkel TA, Erie DA. Direct visualization of asymmetric adenine-nucleotide-induced conformational changes in MutL alpha. Mol Cell 2008; 29:112-21. [PMID: 18206974 DOI: 10.1016/j.molcel.2007.10.030] [Citation(s) in RCA: 107] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2007] [Revised: 08/15/2007] [Accepted: 10/18/2007] [Indexed: 11/26/2022]
Abstract
MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.
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Affiliation(s)
- Elizabeth J Sacho
- Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Ou J, Niessen RC, Lützen A, Sijmons RH, Kleibeuker JH, de Wind N, Rasmussen LJ, Hofstra RMW. Functional analysis helps to clarify the clinical importance of unclassified variants in DNA mismatch repair genes. Hum Mutat 2007; 28:1047-54. [PMID: 17594722 DOI: 10.1002/humu.20580] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome is caused by DNA variations in the DNA mismatch repair (MMR) genes MSH2, MLH1, MSH6, and PMS2. Many of the mutations identified result in premature termination of translation and thus in loss-of-function of the encoded mutated protein. These DNA variations are thought to be pathogenic mutations. However, some patients carry other DNA mutations, referred to as unclassified variants (UVs), which do not lead to such a premature termination of translation; it is not known whether these contribute to the disease phenotype or merely represent rare polymorphisms. This is a major problem which has direct clinical consequences. Several criteria can be used to classify these UVs, such as: whether they segregate with the disease within pedigrees, are absent in control individuals, show a change of amino acid polarity or size, provoke an amino acid change in a domain that is evolutionary conserved and/or shared between proteins belonging to the same protein family, or show altered function in an in vitro assay. In this review we discuss the various functional assays reported for the HNPCC-associated MMR proteins and the outcomes of these tests on UVs identified in patients diagnosed with or suspected of having HNPCC. We conclude that a large proportion of MMR UVs are likely to be pathogenic, suggesting that missense variants of MMR proteins do indeed play a role in HNPCC.
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Affiliation(s)
- Jianghua Ou
- Department of Genetics, University Medical Center Groningen, Groningen, The Netherlands
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40
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Knudsen NØ, Nielsen FC, Vinther L, Bertelsen R, Holten-Andersen S, Liberti SE, Hofstra R, Kooi K, Rasmussen LJ. Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1). Nucleic Acids Res 2007; 35:2609-19. [PMID: 17426132 PMCID: PMC1885640 DOI: 10.1093/nar/gkl1166] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin β/α1,3,7 whereas hMSH2 specifically recognizes importin β/α3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.
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Affiliation(s)
- Nina Østergaard Knudsen
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Finn Cilius Nielsen
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Lena Vinther
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Ronni Bertelsen
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Steen Holten-Andersen
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Sascha Emilie Liberti
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Robert Hofstra
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Krista Kooi
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
| | - Lene Juel Rasmussen
- Department of Science, Systems and Models, Roskilde University, Denmark, Department of Clinical Biochemistry, University Hospital of Copenhagen, Denmark and Department of Medical Genetics, University of Groningen, The Netherlands
- *To whom correspondence should be addressed +45 46742728+45 46 74 30 11
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Bandipalliam P. Variability in the clinical phenotype among families with HNPCC-The potential importance of the location of the mutation in the gene. Int J Cancer 2007; 120:2275-7; author reply 2278. [PMID: 17230503 DOI: 10.1002/ijc.22348] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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42
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McNees CJ, Conlan LA, Tenis N, Heierhorst J. ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage. EMBO J 2005; 24:2447-57. [PMID: 15933716 PMCID: PMC1173145 DOI: 10.1038/sj.emboj.7600704] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2005] [Accepted: 05/10/2005] [Indexed: 12/19/2022] Open
Abstract
Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.
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Affiliation(s)
- Carolyn J McNees
- St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
- Department of Medicine SVH, The University of Melbourne, Fitzroy, Victoria, Australia
| | - Lindus A Conlan
- St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
| | - Nora Tenis
- St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
| | - Jörg Heierhorst
- St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia
- Department of Medicine SVH, The University of Melbourne, Fitzroy, Victoria, Australia
- St Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, VIC 3065, Australia. Tel.: +61 3 9288 2503; Fax: +61 3 9416 2676; E-mail:
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Thompson E, Meldrum CJ, Crooks R, McPhillips M, Thomas L, Spigelman AD, Scott RJ. Hereditary non-polyposis colorectal cancer and the role of hPMS2 and hEXO1 mutations. Clin Genet 2004; 65:215-25. [PMID: 14756672 DOI: 10.1111/j.1399-0004.2004.00214.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant, inherited condition that is characterized primarily by the development of early-onset colorectal cancer and a number of other epithelial malignancies. The underlying genetic basis of the disease is associated with a breakdown of DNA-mismatch repair. There are many genes involved in DNA-mismatch repair, and five of them have been implicated in HNPCC. Two of the genes (hMSH2 and hMLH1) account for the majority of HNPCC families (approximately 60%), and it is not known what the exact contributions of the remaining three genes (hPMS1, hPMS2, and hMSH6) are in relation to this condition. In addition, a sixth gene (hEXO1) has been associated with a disease phenotype that is consistent with HNPCC. Current estimates suggest that all four of these genes, combined, may account for up to 5% of families. In this report, we examine the contribution of hPMS2 and hEXO1 to a well-defined set of families that fulfill the diagnostic criteria for HNPCC. The genes, hPMS2 and hEXO1, were studied by denaturing high performance liquid chromatography (DHPLC) analysis in 21 families that have previously been determined not to have mutations in hMSH2 or hMLH1. hPMS2 accounts for a small proportion of HNPCC families, and none were deemed to be associated with hEXO1. Mutations in hPMS2 appear to account for a small proportion of families adhering to the Amsterdam II criteria, whereas hEXO1 does not appear to be associated with HNPCC.
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Affiliation(s)
- E Thompson
- Discipline of Medical Genetics, Faculty of Health, University of Newcastle and Hunter Medical Research Institute, Newcastle, NSW, Australia
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Lützen A, Liberti SE, Rasmussen LJ. Cadmium inhibits human DNA mismatch repair in vivo. Biochem Biophys Res Commun 2004; 321:21-5. [PMID: 15358209 DOI: 10.1016/j.bbrc.2004.06.102] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2004] [Indexed: 10/26/2022]
Abstract
The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.
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Affiliation(s)
- Anne Lützen
- Department of Life Sciences and Chemistry, Roskilde University, DK-4000 Roskilde, Denmark
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Abstract
We have developed a purified system that supports mismatch-dependent 5'-->3' excision. In the presence of RPA, ATP, and a mismatch, MutSalpha activates 5'-->3' excision by EXOI, and excision terminates after removal of the mispair. MutSalpha confers high processivity on EXOI, and termination is due to RPA-dependent displacement of this processive complex from the helix and a weak ability of EXOI to reload at the RPA-bound gap in the product, as well as MutSalpha- and MutLalpha-dependent suppression of EXOI activity in the absence of a mismatch cofactor. As observed in the purified system, excision directed by a 5' strand break in HeLa nuclear extract can proceed in the absence of MutLalpha or PCNA, although 3' excision in the extract system requires both proteins.
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Affiliation(s)
- Jochen Genschel
- Department of Biochemistry, Box 3711, Duke University Medical Center, Durham, NC 27710, USA
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47
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Nielsen FC, Jäger AC, Lützen A, Bundgaard JR, Rasmussen LJ. Characterization of human exonuclease 1 in complex with mismatch repair proteins, subcellular localization and association with PCNA. Oncogene 2003; 23:1457-68. [PMID: 14676842 DOI: 10.1038/sj.onc.1207265] [Citation(s) in RCA: 63] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Human exonuclease 1 (hEXO1) has been implicated in DNA mismatch repair (MMR), replication, and recombination, but the nature of its interaction with these cellular processes is still ambiguous. We show that hEXO1 colocalizes with proliferating cell nuclear antigen (PCNA) at DNA replication sites and that the C-terminal region of hEXO1 is sufficient for this localization. We also show that both hMLH1-hPMS2 (MutLalpha) and hMLH1-hEXO1 complexes are formed in a reaction mixture containing all three proteins. Moreover, hEXO1 5' double-stranded exonuclease activity on a homoduplex substrate but not on a substrate containing a G/T mismatch was inhibited by complex formation with hMSH2-hMSH6 (MutSalpha) or MutLalpha. Taken together, the results support a model in which hEXO1 plays a role in events at the replication sites as well as a functional role in the MMR and/or recombination processes.
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Affiliation(s)
- Finn Cilius Nielsen
- Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark
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Hansen LT, Thykjaer T, Ørntoft TF, Rasmussen LJ, Keller P, Spang-Thomsen M, Edmonston TB, Schmutte C, Fishel R, Petersen LN. The role of mismatch repair in small-cell lung cancer cells. Eur J Cancer 2003; 39:1456-67. [PMID: 12826050 DOI: 10.1016/s0959-8049(03)00306-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The role of mismatch repair (MMR) in small-cell lung cancer (SCLC) is controversial, as the phenotype of a MMR-deficiency, microsatellite instability (MSI), has been reported to range from 0 to 76%. We studied the MMR pathway in a panel of 21 SCLC cell lines and observed a highly heterogeneous pattern of MMR gene expression. A significant correlation between the mRNA and protein levels was found. We demonstrate that low hMLH1 gene expression was not linked to promoter CpG methylation. One cell line (86MI) was found to be deficient in MMR and exhibited resistance to the alkylating agent MNNG. Surprisingly, MSI was not detected in 86MI and it appears to express all the major MMR components hMSH2, hMSH6, hMLH1, hPMS2, hMSH3, hMLH3, MBD4 (MED1) and hExo1. These data are consistent with at least two possibilities: (1) A missense mutation in one of the MMR genes, which dissociates MSI from drug resistance, or (2) inactivation of a second pathway that leads to MMR-deficiency and MNNG resistance, but induces negligible levels of MSI. We conclude that MMR deficiency is largely not associated with the pathogenesis of SCLC.
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Affiliation(s)
- L T Hansen
- Institute of Molecular Pathology, University of Copenhagen, DK-2100 Copenhagen, Denmark.
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Abstract
DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.
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Affiliation(s)
- Xiaosheng Wu
- Transplantation Biology, Mayo Clinic, Rochester, Minnesota 55905, USA
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Ladd PD, Wilson DM, Kelley MR, Skalnik DG. Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. DNA Repair (Amst) 2003; 2:187-98. [PMID: 12531389 DOI: 10.1016/s1568-7864(02)00195-7] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5' to 3' exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.
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Affiliation(s)
- Paula D Ladd
- Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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