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Bhaumik SR. Active genome integrity. DNA Repair (Amst) 2025; 146:103816. [PMID: 39921947 DOI: 10.1016/j.dnarep.2025.103816] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/10/2025]
Affiliation(s)
- Sukesh R Bhaumik
- Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale IL-62901, USA.
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2
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Kutz J, Schmietendorf H, Rahman SA, Opel F, Pospiech H. HROB Is Implicated in DNA Replication. Genes (Basel) 2024; 15:1587. [PMID: 39766854 PMCID: PMC11675949 DOI: 10.3390/genes15121587] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2024] [Revised: 11/29/2024] [Accepted: 12/05/2024] [Indexed: 01/11/2025] Open
Abstract
DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair. We identified HROB independently as a nuclear protein whose expression is co-regulated with various DNA replication factors. Accordingly, the HROB protein level showed a maximum in S phase and a downregulation in quiescence. Structural prediction and homology searches revealed that HROB is a largely intrinsically disordered protein bearing a helix-rich region and a canonical oligonucleotide/oligosaccharide-binding-fold motif that originated early in eukaryotic evolution. Employing a flow cytometry Förster resonance energy transfer (FRET) assay, we detected associations between HROB and proteins of the DNA replication machinery. Moreover, ectopic expression of HROB protein led to an almost complete shutdown of DNA replication. The available data imply a function for HROB during DNA replication across barriers such as ICLs.
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Affiliation(s)
- Julia Kutz
- Project Group Biochemistry, Leibniz Institute on Aging—Fritz Lipmann Institute, D-07745 Jena, Germany; (J.K.); (H.S.); (S.A.R.); (F.O.)
- Institute of Biochemistry and Biophysics, Faculty of Biological Sciences, Friedrich Schiller University, D-07745 Jena, Germany
| | - Hannes Schmietendorf
- Project Group Biochemistry, Leibniz Institute on Aging—Fritz Lipmann Institute, D-07745 Jena, Germany; (J.K.); (H.S.); (S.A.R.); (F.O.)
- Institute of Biochemistry and Biophysics, Faculty of Biological Sciences, Friedrich Schiller University, D-07745 Jena, Germany
| | - Sheikh Anika Rahman
- Project Group Biochemistry, Leibniz Institute on Aging—Fritz Lipmann Institute, D-07745 Jena, Germany; (J.K.); (H.S.); (S.A.R.); (F.O.)
- Institute of Biochemistry and Biophysics, Faculty of Biological Sciences, Friedrich Schiller University, D-07745 Jena, Germany
| | - Franz Opel
- Project Group Biochemistry, Leibniz Institute on Aging—Fritz Lipmann Institute, D-07745 Jena, Germany; (J.K.); (H.S.); (S.A.R.); (F.O.)
- Department of Medical Engineering and Biotechnology, Ernst-Abbe University of Applied Sciences, D-07745 Jena, Germany
| | - Helmut Pospiech
- Project Group Biochemistry, Leibniz Institute on Aging—Fritz Lipmann Institute, D-07745 Jena, Germany; (J.K.); (H.S.); (S.A.R.); (F.O.)
- Institute of Biochemistry and Biophysics, Faculty of Biological Sciences, Friedrich Schiller University, D-07745 Jena, Germany
- Department of Obstetrics and Gynecology, University Hospital Düsseldorf and Heinrich-Heine University, D-40225 Düsseldorf, Germany
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3
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de Moura A, Karschau J. Mathematical model for the distribution of DNA replication origins. Phys Rev E 2024; 110:034408. [PMID: 39425392 DOI: 10.1103/physreve.110.034408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Accepted: 09/03/2024] [Indexed: 10/21/2024]
Abstract
DNA replication in yeast and in many other organisms starts from well-defined locations on the DNA known as replication origins. The spatial distribution of these origins in the genome is particularly important in ensuring that replication is completed quickly. Cells are more vulnerable to DNA damage and other forms of stress while they are replicating their genome. This raises the possibility that the spatial distribution of origins is under selection pressure. In this paper we investigate the hypothesis that natural selection favors origin distributions leading to shorter replication times. Using a simple mathematical model, we show that this hypothesis leads to two main predictions about the origin distributions: that neighboring origins that are inefficient (less likely to fire) are more likely to be close to each other than efficient origins; and that neighboring origins with larger differences in firing times are more likely to be close to each other than origins with similar firing times. We test these predictions using next-generation sequencing data, and show that they are both supported by the data.
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4
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Chauhan AS, Jhujh SS, Stewart GS. E3 ligases: a ubiquitous link between DNA repair, DNA replication and human disease. Biochem J 2024; 481:923-944. [PMID: 38985307 PMCID: PMC11346458 DOI: 10.1042/bcj20240124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Revised: 05/20/2024] [Accepted: 05/24/2024] [Indexed: 07/11/2024]
Abstract
Maintenance of genome stability is of paramount importance for the survival of an organism. However, genomic integrity is constantly being challenged by various endogenous and exogenous processes that damage DNA. Therefore, cells are heavily reliant on DNA repair pathways that have evolved to deal with every type of genotoxic insult that threatens to compromise genome stability. Notably, inherited mutations in genes encoding proteins involved in these protective pathways trigger the onset of disease that is driven by chromosome instability e.g. neurodevelopmental abnormalities, neurodegeneration, premature ageing, immunodeficiency and cancer development. The ability of cells to regulate the recruitment of specific DNA repair proteins to sites of DNA damage is extremely complex but is primarily mediated by protein post-translational modifications (PTMs). Ubiquitylation is one such PTM, which controls genome stability by regulating protein localisation, protein turnover, protein-protein interactions and intra-cellular signalling. Over the past two decades, numerous ubiquitin (Ub) E3 ligases have been identified to play a crucial role not only in the initiation of DNA replication and DNA damage repair but also in the efficient termination of these processes. In this review, we discuss our current understanding of how different Ub E3 ligases (RNF168, TRAIP, HUWE1, TRIP12, FANCL, BRCA1, RFWD3) function to regulate DNA repair and replication and the pathological consequences arising from inheriting deleterious mutations that compromise the Ub-dependent DNA damage response.
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Affiliation(s)
- Anoop S. Chauhan
- Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, U.K
| | - Satpal S. Jhujh
- Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, U.K
| | - Grant S. Stewart
- Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, U.K
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5
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Reed TT, Kendal AH, Wozniak KJ, Simmons LA. DNA replication initiation timing is important for maintaining genome integrity. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.06.18.599555. [PMID: 38948856 PMCID: PMC11212987 DOI: 10.1101/2024.06.18.599555] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/02/2024]
Abstract
DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that ΔccrZ and several ccrZ alleles under-initiate DNA replication while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.
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Affiliation(s)
- Tristan T. Reed
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
| | - Abigail H. Kendal
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
| | - Katherine J Wozniak
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
- Present address: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030
| | - Lyle A. Simmons
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
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6
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Hashimoto Y, Sadano K, Miyata N, Ito H, Tanaka H. Novel role of DONSON in CMG helicase assembly during vertebrate DNA replication initiation. EMBO J 2023; 42:e114131. [PMID: 37458194 PMCID: PMC10476173 DOI: 10.15252/embj.2023114131] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Revised: 05/27/2023] [Accepted: 06/29/2023] [Indexed: 09/05/2023] Open
Abstract
CMG (Cdc45-MCM-GINS) helicase assembly at the replication origin is the culmination of eukaryotic DNA replication initiation. This process can be reconstructed in vitro using defined factors in Saccharomyces cerevisiae; however, in vertebrates, origin-dependent CMG formation has not yet been achieved partly due to the lack of a complete set of known initiator proteins. Since a microcephaly gene product, DONSON, was reported to remodel the CMG helicase under replication stress, we analyzed its role in DNA replication using a Xenopus cell-free system. We found that DONSON was essential for the replisome assembly. In vertebrates, DONSON physically interacted with GINS and Polε via its conserved N-terminal PGY and NPF motifs, and the DONSON-GINS interaction contributed to the replisome assembly. DONSON's chromatin association during replication initiation required the pre-replicative complex, TopBP1, and kinase activities of S-CDK and DDK. Both S-CDK and DDK required DONSON to trigger replication initiation. Moreover, human DONSON could substitute for the Xenopus protein in a cell-free system. These findings indicate that vertebrate DONSON is a novel initiator protein essential for CMG helicase assembly.
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Affiliation(s)
- Yoshitami Hashimoto
- School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
| | - Kota Sadano
- School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
| | - Nene Miyata
- School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
| | - Haruka Ito
- School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
| | - Hirofumi Tanaka
- School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
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7
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Brison O, Gnan S, Azar D, Koundrioukoff S, Melendez-Garcia R, Kim SJ, Schmidt M, El-Hilali S, Jaszczyszyn Y, Lachages AM, Thermes C, Chen CL, Debatisse M. Mistimed origin licensing and activation stabilize common fragile sites under tight DNA-replication checkpoint activation. Nat Struct Mol Biol 2023; 30:539-550. [PMID: 37024657 DOI: 10.1038/s41594-023-00949-1] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2021] [Accepted: 02/28/2023] [Indexed: 04/08/2023]
Abstract
Genome integrity requires replication to be completed before chromosome segregation. The DNA-replication checkpoint (DRC) contributes to this coordination by inhibiting CDK1, which delays mitotic onset. Under-replication of common fragile sites (CFSs), however, escapes surveillance, resulting in mitotic chromosome breaks. Here we asked whether loose DRC activation induced by modest stresses commonly used to destabilize CFSs could explain this leakage. We found that tightening DRC activation or CDK1 inhibition stabilizes CFSs in human cells. Repli-Seq and molecular combing analyses showed a burst of replication initiations implemented in mid S-phase across a subset of late-replicating sequences, including CFSs, while the bulk genome was unaffected. CFS rescue and extra-initiations required CDC6 and CDT1 availability in S-phase, implying that CDK1 inhibition permits mistimed origin licensing and firing. In addition to delaying mitotic onset, tight DRC activation therefore supports replication completion of late origin-poor domains at risk of under-replication, two complementary roles preserving genome stability.
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Affiliation(s)
- Olivier Brison
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France
- Paris-Saclay University, Gif-sur-Yvette, France
| | - Stefano Gnan
- Curie Institute, PSL Research University, CNRS UMR 3244, Paris, France
- Sorbonne University, Paris, France
| | - Dana Azar
- Curie Institute, PSL Research University, CNRS UMR 3244, Paris, France
- Sorbonne University, Paris, France
- Laboratoire Biodiversité et Génomique Fonctionnelle, Faculté des Sciences, Université Saint-Joseph, Beirut, Lebanon
| | - Stéphane Koundrioukoff
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France
- Sorbonne University, Paris, France
| | - Rodrigo Melendez-Garcia
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France
- Paris-Saclay University, Gif-sur-Yvette, France
| | - Su-Jung Kim
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France
- Paris-Saclay University, Gif-sur-Yvette, France
| | - Mélanie Schmidt
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France
- Paris-Saclay University, Gif-sur-Yvette, France
| | - Sami El-Hilali
- Curie Institute, PSL Research University, CNRS UMR 3244, Paris, France
- Sorbonne University, Paris, France
- Villefranche sur mer Developmental Biology Laboratory, CNRS UMR7009, Villefranche-sur-Mer, France
| | - Yan Jaszczyszyn
- Paris-Saclay University, Gif-sur-Yvette, France
- Institute for Integrative Biology of the Cell (I2BC), UMR 9198CNRS, CEA, Paris-Sud University, Gif-sur-Yvette, France
| | - Anne-Marie Lachages
- Curie Institute, PSL Research University, CNRS UMR 3244, Paris, France
- UTCBS, CNRS UMR 8258/ INSERM U 1267, Sorbonne-Paris-Cité University, Paris, France
| | - Claude Thermes
- Paris-Saclay University, Gif-sur-Yvette, France
- Institute for Integrative Biology of the Cell (I2BC), UMR 9198CNRS, CEA, Paris-Sud University, Gif-sur-Yvette, France
| | - Chun-Long Chen
- Curie Institute, PSL Research University, CNRS UMR 3244, Paris, France
- Sorbonne University, Paris, France
| | - Michelle Debatisse
- CNRS UMR 9019, Gustave Roussy Institute, Villejuif, France.
- Sorbonne University, Paris, France.
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8
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Amin A, Wu R, Khan MA, Cheung MH, Liang Y, Liu C, Zhu G, Yu ZL, Liang C. An essential Noc3p dimerization cycle mediates ORC double-hexamer formation in replication licensing. Life Sci Alliance 2023; 6:e202201594. [PMID: 36599624 PMCID: PMC9813392 DOI: 10.26508/lsa.202201594] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2022] [Revised: 12/17/2022] [Accepted: 12/19/2022] [Indexed: 01/05/2023] Open
Abstract
Replication licensing, a prerequisite of DNA replication, helps to ensure once-per-cell-cycle genome duplication. Some DNA replication-initiation proteins are sequentially loaded onto replication origins to form pre-replicative complexes (pre-RCs). ORC and Noc3p bind replication origins throughout the cell cycle, providing a platform for pre-RC assembly. We previously reported that cell cycle-dependent ORC dimerization is essential for the chromatin loading of the symmetric MCM double-hexamers. Here, we used Saccharomyces cerevisiae separation-of-function NOC3 mutants to confirm the separable roles of Noc3p in DNA replication and ribosome biogenesis. We also show that an essential and cell cycle-dependent Noc3p dimerization cycle regulates the ORC dimerization cycle. Noc3p dimerizes at the M-to-G1 transition and de-dimerizes in S-phase. The Noc3p dimerization cycle coupled with the ORC dimerization cycle enables replication licensing, protects nascent sister replication origins after replication initiation, and prevents re-replication. This study has revealed a new mechanism of replication licensing and elucidated the molecular mechanism of Noc3p as a mediator of ORC dimerization in pre-RC formation.
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Affiliation(s)
- Aftab Amin
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
- School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China
| | - Rentian Wu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Muhammad Ajmal Khan
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Man Hei Cheung
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Yanting Liang
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Changdong Liu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Guang Zhu
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
| | - Zhi-Ling Yu
- School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China
| | - Chun Liang
- Division of Life Science, Center for Cancer Research, and State Key Lab of Molecular Neuroscience, Hong Kong University of Science and Technology, Hong Kong, China
- EnKang Pharmaceuticals (Guangzhou), Ltd., Guangzhou, China
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9
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Construction of a simple, localized and homogeneous fluorescence detection platform for T4 PNK activity based on tetrahedral DNA nanostructure-mediated primer exchange reaction. Microchem J 2022. [DOI: 10.1016/j.microc.2022.107989] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
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10
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Martín-Rufo R, de la Vega-Barranco G, Lecona E. Ubiquitin and SUMO as timers during DNA replication. Semin Cell Dev Biol 2022; 132:62-73. [PMID: 35210137 DOI: 10.1016/j.semcdb.2022.02.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2021] [Revised: 02/12/2022] [Accepted: 02/14/2022] [Indexed: 12/14/2022]
Abstract
Every time a cell copies its DNA the genetic material is exposed to the acquisition of mutations and genomic alterations that corrupt the information passed on to daughter cells. A tight temporal regulation of DNA replication is necessary to ensure the full copy of the DNA while preventing the appearance of genomic instability. Protein modification by ubiquitin and SUMO constitutes a very complex and versatile system that allows the coordinated control of protein stability, activity and interactome. In chromatin, their action is complemented by the AAA+ ATPase VCP/p97 that recognizes and removes ubiquitylated and SUMOylated factors from specific cellular compartments. The concerted action of the ubiquitin/SUMO system and VCP/p97 determines every step of DNA replication enforcing the ordered activation/inactivation, loading/unloading and stabilization/destabilization of replication factors. Here we analyze the mechanisms used by ubiquitin/SUMO and VCP/p97 to establish molecular timers throughout DNA replication and their relevance in maintaining genome stability. We propose that these PTMs are the main molecular watch of DNA replication from origin recognition to replisome disassembly.
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Affiliation(s)
- Rodrigo Martín-Rufo
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Guillermo de la Vega-Barranco
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain
| | - Emilio Lecona
- Chromatin, Cancer and the Ubiquitin System lab, Centre for Molecular Biology Severo Ochoa (CBMSO, CSIC-UAM), Department of Genome Dynamics and Function, Madrid 28049, Spain.
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11
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Transcriptome Analysis of the Anti-Proliferative Effects of Ginsenoside Rh3 on HCT116 Colorectal Cancer Cells. MOLECULES (BASEL, SWITZERLAND) 2022; 27:molecules27155002. [PMID: 35956952 PMCID: PMC9370307 DOI: 10.3390/molecules27155002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 07/27/2022] [Accepted: 08/03/2022] [Indexed: 11/16/2022]
Abstract
The mechanism of ginsenoside Rh3 activity against cancer remains unclear. This study aimed to investigate the underlying mechanism. The effects of Rh3 on the cell proliferation, migration and invasion, and cycle and apoptosis were analyzed using CCK-8 assay, transwell migration assay and flow cytometry, respectively. The RNA transcriptome was sequenced and data were analyzed by R software. Protein expression and protein-protein interactions were determined by Western blotting and co-immunoprecipitation, respectively. The results showed Rh3 inhibited HCT116 cell proliferation, invasion, and migration, arrested cells at G1 phase; and increased apoptosis. Rh3 downregulated 314 genes and upregulated 371 genes. Gene Set Enrichment Analysis (GSEA) using The Kyoto Encyclopedia of Genes Genomics ranked DNA replication first, while GSEA using Gene Ontology ranked the initiation of DNA replication first. Compared with tumor data from The Cancer Genome Atlas (TCGA), most of genes related to DNA replication were oppositely regulated by Rh3. Furthermore, Rh3 down-regulated key protein expression related to DNA replication (Orc6, Cdt1, and Mcm2), but did not affect the loading of Mcm complexes onto ORC complexes nor the phosphorylation at ser139 of Mcm2. Therefore, Rh3 may inhibit colorectal cancer HCT116 cells by downregulation of genes related to DNA replication.
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12
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Li Y, Hartemink AJ, MacAlpine DM. Cell-Cycle-Dependent Chromatin Dynamics at Replication Origins. Genes (Basel) 2021; 12:genes12121998. [PMID: 34946946 PMCID: PMC8701747 DOI: 10.3390/genes12121998] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 12/02/2021] [Accepted: 12/08/2021] [Indexed: 01/20/2023] Open
Abstract
Origins of DNA replication are specified by the ordered recruitment of replication factors in a cell-cycle–dependent manner. The assembly of the pre-replicative complex in G1 and the pre-initiation complex prior to activation in S phase are well characterized; however, the interplay between the assembly of these complexes and the local chromatin environment is less well understood. To investigate the dynamic changes in chromatin organization at and surrounding replication origins, we used micrococcal nuclease (MNase) to generate genome-wide chromatin occupancy profiles of nucleosomes, transcription factors, and replication proteins through consecutive cell cycles in Saccharomyces cerevisiae. During each G1 phase of two consecutive cell cycles, we observed the downstream repositioning of the origin-proximal +1 nucleosome and an increase in protected DNA fragments spanning the ARS consensus sequence (ACS) indicative of pre-RC assembly. We also found that the strongest correlation between chromatin occupancy at the ACS and origin efficiency occurred in early S phase, consistent with the rate-limiting formation of the Cdc45–Mcm2-7–GINS (CMG) complex being a determinant of origin activity. Finally, we observed nucleosome disruption and disorganization emanating from replication origins and traveling with the elongating replication forks across the genome in S phase, likely reflecting the disassembly and assembly of chromatin ahead of and behind the replication fork, respectively. These results provide insights into cell-cycle–regulated chromatin dynamics and how they relate to the regulation of origin activity.
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Affiliation(s)
- Yulong Li
- Department of Computer Science, Duke University, Durham, NC 27708, USA;
| | - Alexander J. Hartemink
- Department of Computer Science, Duke University, Durham, NC 27708, USA;
- Correspondence: (A.J.H.); (D.M.M.)
| | - David M. MacAlpine
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
- Correspondence: (A.J.H.); (D.M.M.)
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13
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Dolson A, Sauty SM, Shaban K, Yankulov K. Dbf4-Dependent Kinase: DDK-ated to post-initiation events in DNA replication. Cell Cycle 2021; 20:2348-2360. [PMID: 34662256 DOI: 10.1080/15384101.2021.1986999] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Dbf4-Dependent Kinase (DDK) has a well-established essential role at origins of DNA replication, where it phosphorylates and activates the replicative MCM helicase. It also acts in the response to mutagens and in DNA repair as well as in key steps during meiosis. Recent studies have indicated that, in addition to the MCM helicase, DDK phosphorylates several substrates during the elongation stage of DNA replication or upon replication stress. However, these activities of DDK are not essential for viability. Dbf4-Dependent Kinase is also emerging as a key factor in the regulation of genome-wide origin firing and in replication-coupled chromatin assembly. In this review, we summarize recent progress in our understanding of the diverse roles of DDK.
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Affiliation(s)
- Andrew Dolson
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| | - Safia Mahabub Sauty
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| | - Kholoud Shaban
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
| | - Krassimir Yankulov
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
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