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1
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Madhani HD. Mechanisms of Inheritance of Chromatin States: From Yeast to Human. Annu Rev Biophys 2025; 54:59-79. [PMID: 39715046 DOI: 10.1146/annurev-biophys-070524-091904] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2024]
Abstract
In this article I review mechanisms that underpin epigenetic inheritance of CpG methylation and histone H3 lysine 9 methylation (H3K9me) in chromatin in fungi and mammals. CpG methylation can be faithfully inherited epigenetically at some sites for a lifetime in vertebrates and, remarkably, can be propagated for millions of years in some fungal lineages. Transmission of methylation patterns requires maintenance-type DNA methyltransferases (DNMTs) that recognize hemimethylated CpG DNA produced by replication. DNMT1 is the maintenance enzyme in vertebrates; we recently identified DNMT5 as an ATP-dependent CpG maintenance enzyme found in fungi and protists. In vivo, CpG methylation is coupled to H3K9me. H3K9me is itself reestablished after replication via local histone H3-H4 tetramer recycling involving mobile and nonmobile chaperones, de novo nucleosome assembly, and read-write mechanisms that modify naive nucleosomes. Additional proteins recognize hemimethylated CpG or fully methylated CpG-containing motifs and enhance restoration of methylation by recruiting and/or activating the maintenance methylase.
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Affiliation(s)
- Hiten D Madhani
- Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA;
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2
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Song A, Wang Y, Liu C, Yu J, Zhang Z, Lan L, Lin H, Zhao J, Li G. Replication-coupled inheritance of chromatin states. CELL INSIGHT 2024; 3:100195. [PMID: 39391004 PMCID: PMC11462216 DOI: 10.1016/j.cellin.2024.100195] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/05/2024] [Revised: 08/05/2024] [Accepted: 08/05/2024] [Indexed: 10/12/2024]
Abstract
During the development of eukaryote, faithful inheritance of chromatin states is central to the maintenance of cell fate. DNA replication poses a significant challenge for chromatin state inheritance because every nucleosome in the genome is disrupted as the replication fork passes. It has been found that many factors including DNA polymerases, histone chaperones, as well as, RNA Pol II and histone modifying enzymes coordinate spatially and temporally to maintain the epigenome during this progress. In this review, we provide a summary of the detailed mechanisms of replication-coupled nucleosome assembly and post-replication chromatin maturation, highlight the inheritance of chromatin states and epigenome during these processes, and discuss the future directions and challenges in this field.
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Affiliation(s)
- Aoqun Song
- New Cornerstone Science Laboratory, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Yunting Wang
- New Cornerstone Science Laboratory, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China
| | - Cuifang Liu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
| | - Juan Yu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zixu Zhang
- New Cornerstone Science Laboratory, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Liting Lan
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Haiyan Lin
- New Cornerstone Science Laboratory, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
| | - Jicheng Zhao
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
- Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, College of Medicine, Qingdao University, Qingdao, 266021, China
| | - Guohong Li
- New Cornerstone Science Laboratory, Frontier Science Center for Immunology and Metabolism, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, 430072, China
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- Key Laboratory of Epigenetic Regulation and Intervention, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
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3
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Yu J, Zhang Y, Fang Y, Paulo JA, Yaghoubi D, Hua X, Shipkovenska G, Toda T, Zhang Z, Gygi SP, Jia S, Li Q, Moazed D. A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance. Cell 2024; 187:5010-5028.e24. [PMID: 39094570 PMCID: PMC11380579 DOI: 10.1016/j.cell.2024.07.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2023] [Revised: 03/17/2024] [Accepted: 07/03/2024] [Indexed: 08/04/2024]
Abstract
Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.
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Affiliation(s)
- Juntao Yu
- Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Yujie Zhang
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
| | - Yimeng Fang
- Department of Biological Sciences, Columbia University, New York, NY 10027, USA
| | - Joao A Paulo
- Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Dadmehr Yaghoubi
- Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Xu Hua
- Institute for Cancer Genetics, Department of Pediatrics, and Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Gergana Shipkovenska
- Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Takenori Toda
- Department of Biological Sciences, Columbia University, New York, NY 10027, USA
| | - Zhiguo Zhang
- Institute for Cancer Genetics, Department of Pediatrics, and Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY 10032, USA
| | - Steven P Gygi
- Department of Cell Biology, Harvard Medical School, Boston, MA, USA
| | - Songtao Jia
- Department of Biological Sciences, Columbia University, New York, NY 10027, USA
| | - Qing Li
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
| | - Danesh Moazed
- Howard Hughes Medical Institute, Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
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4
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Madhani HD. Deep learning meets histones at the replication fork. Cell 2024; 187:4824-4826. [PMID: 39241742 DOI: 10.1016/j.cell.2024.07.055] [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: 07/29/2024] [Revised: 07/30/2024] [Accepted: 07/30/2024] [Indexed: 09/09/2024]
Abstract
Epigenetic inheritance of heterochromatin requires transfer of parental H3-H4 tetramers to both daughter duplexes during replication. Three recent papers exploit yeast genetics coupled to inheritance assays and AlphaFold2-multimer predictions coupled to biochemistry to reveal that a replisome component (Mrc1/CLASPIN) is an H3-H4 tetramer chaperone important for parental histone transfer to daughters.
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Affiliation(s)
- Hiten D Madhani
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
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5
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You Z, Masai H. Assembly, Activation, and Helicase Actions of MCM2-7: Transition from Inactive MCM2-7 Double Hexamers to Active Replication Forks. BIOLOGY 2024; 13:629. [PMID: 39194567 DOI: 10.3390/biology13080629] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/21/2024] [Revised: 08/13/2024] [Accepted: 08/14/2024] [Indexed: 08/29/2024]
Abstract
In this review, we summarize the processes of the assembly of multi-protein replisomes at the origins of replication. Replication licensing, the loading of inactive minichromosome maintenance double hexamers (dhMCM2-7) during the G1 phase, is followed by origin firing triggered by two serine-threonine kinases, Cdc7 (DDK) and CDK, leading to the assembly and activation of Cdc45/MCM2-7/GINS (CMG) helicases at the entry into the S phase and the formation of replisomes for bidirectional DNA synthesis. Biochemical and structural analyses of the recruitment of initiation or firing factors to the dhMCM2-7 for the formation of an active helicase and those of origin melting and DNA unwinding support the steric exclusion unwinding model of the CMG helicase.
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Affiliation(s)
- Zhiying You
- Genome Dynamics Project, Department of Basic Medical Sciences, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan
| | - Hisao Masai
- Genome Dynamics Project, Department of Basic Medical Sciences, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8561, Japan
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6
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Du W, Shi G, Shan CM, Li Z, Zhu B, Jia S, Li Q, Zhang Z. Mechanisms of chromatin-based epigenetic inheritance. SCIENCE CHINA. LIFE SCIENCES 2022; 65:2162-2190. [PMID: 35792957 PMCID: PMC10311375 DOI: 10.1007/s11427-022-2120-1] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/09/2022] [Accepted: 04/27/2022] [Indexed: 06/15/2023]
Abstract
Multi-cellular organisms such as humans contain hundreds of cell types that share the same genetic information (DNA sequences), and yet have different cellular traits and functions. While how genetic information is passed through generations has been extensively characterized, it remains largely obscure how epigenetic information encoded by chromatin regulates the passage of certain traits, gene expression states and cell identity during mitotic cell divisions, and even through meiosis. In this review, we will summarize the recent advances on molecular mechanisms of epigenetic inheritance, discuss the potential impacts of epigenetic inheritance during normal development and in some disease conditions, and outline future research directions for this challenging, but exciting field.
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Affiliation(s)
- Wenlong Du
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Guojun Shi
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
| | - Chun-Min Shan
- State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zhiming Li
- Institutes of Cancer Genetics, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, 10032, USA
| | - Bing Zhu
- National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China.
| | - Songtao Jia
- Department of Biological Sciences, Columbia University, New York, NY, 10027, USA.
| | - Qing Li
- State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences and Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China.
| | - Zhiguo Zhang
- Institutes of Cancer Genetics, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY, 10032, USA.
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7
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Evrin C, Serra‐Cardona A, Duan S, Mukherjee PP, Zhang Z, Labib KPM. Spt5 histone binding activity preserves chromatin during transcription by RNA polymerase II. EMBO J 2022; 41:e109783. [PMID: 35102600 PMCID: PMC8886531 DOI: 10.15252/embj.2021109783] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2021] [Revised: 12/22/2021] [Accepted: 01/06/2022] [Indexed: 12/30/2022] Open
Abstract
Nucleosomes are disrupted transiently during eukaryotic transcription, yet the displaced histones must be retained and redeposited onto DNA, to preserve nucleosome density and associated histone modifications. Here, we show that the essential Spt5 processivity factor of RNA polymerase II (Pol II) plays a direct role in this process in budding yeast. Functional orthologues of eukaryotic Spt5 are present in archaea and bacteria, reflecting its universal role in RNA polymerase processivity. However, eukaryotic Spt5 is unique in having an acidic amino terminal tail (Spt5N) that is sandwiched between the downstream nucleosome and the upstream DNA that emerges from Pol II. We show that Spt5N contains a histone-binding motif that is required for viability in yeast cells and prevents loss of nucleosomal histones within actively transcribed regions. These findings indicate that eukaryotic Spt5 combines two essential activities, which together couple processive transcription to the efficient capture and re-deposition of nucleosomal histones.
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Affiliation(s)
- Cecile Evrin
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Albert Serra‐Cardona
- Institute for Cancer GeneticsDepartment of Pediatrics and Department of Genetics and DevelopmentColumbia University Irving Medical CenterNew YorkNYUSA
| | - Shoufu Duan
- Institute for Cancer GeneticsDepartment of Pediatrics and Department of Genetics and DevelopmentColumbia University Irving Medical CenterNew YorkNYUSA
| | - Progya P Mukherjee
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
| | - Zhiguo Zhang
- Institute for Cancer GeneticsDepartment of Pediatrics and Department of Genetics and DevelopmentColumbia University Irving Medical CenterNew YorkNYUSA
| | - Karim P M Labib
- The MRC Protein Phosphorylation and Ubiquitylation UnitSchool of Life SciencesUniversity of DundeeDundeeUK
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Santos-Rosa H, Millán-Zambrano G, Han N, Leonardi T, Klimontova M, Nasiscionyte S, Pandolfini L, Tzelepis K, Bartke T, Kouzarides T. Methylation of histone H3 at lysine 37 by Set1 and Set2 prevents spurious DNA replication. Mol Cell 2021; 81:2793-2807.e8. [PMID: 33979575 PMCID: PMC7612968 DOI: 10.1016/j.molcel.2021.04.021] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 02/09/2021] [Accepted: 04/21/2021] [Indexed: 11/22/2022]
Abstract
DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae, share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication.
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Affiliation(s)
- Helena Santos-Rosa
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
| | - Gonzalo Millán-Zambrano
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), 41092 Sevilla, Spain
| | - Namshik Han
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Milner Therapeutics Institute, University of Cambridge, Cambridge CB2 0AW, UK
| | - Tommaso Leonardi
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Center for Genomic Science Istituto Italiano di Tecnologia (IIT), 20139 Milano, Italy
| | - Marie Klimontova
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK
| | - Simona Nasiscionyte
- Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Luca Pandolfini
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Istituto Italiano di Tecnologia (IIT), Center for Human Technologies (CHT), 16152 Genova, Italy
| | - Kostantinos Tzelepis
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK; Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK
| | - Till Bartke
- Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany
| | - Tony Kouzarides
- The Gurdon Institute and Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
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9
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Wang Z, Wang W, Wang L. Epigenetic regulation of covalently closed circular DNA minichromosome in hepatitis B virus infection. BIOPHYSICS REPORTS 2020. [DOI: 10.1007/s41048-020-00112-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
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10
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Leturcq M, Mortuaire M, Hardivillé S, Schulz C, Lefebvre T, Vercoutter-Edouart AS. O-GlcNAc transferase associates with the MCM2-7 complex and its silencing destabilizes MCM-MCM interactions. Cell Mol Life Sci 2018; 75:4321-4339. [PMID: 30069701 PMCID: PMC6208770 DOI: 10.1007/s00018-018-2874-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2018] [Revised: 07/06/2018] [Accepted: 07/13/2018] [Indexed: 02/07/2023]
Abstract
O-GlcNAcylation of proteins is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). The homeostasis of O-GlcNAc cycling is regulated during cell cycle progression and is essential for proper cellular division. We previously reported the O-GlcNAcylation of the minichromosome maintenance proteins MCM2, MCM3, MCM6 and MCM7. These proteins belong to the MCM2-7 complex which is crucial for the initiation of DNA replication through its DNA helicase activity. Here we show that the six subunits of MCM2-7 are O-GlcNAcylated and that O-GlcNAcylation of MCM proteins mainly occurs in the chromatin-bound fraction of synchronized human cells. Moreover, we identify stable interaction between OGT and several MCM subunits. We also show that down-regulation of OGT decreases the chromatin binding of MCM2, MCM6 and MCM7 without affecting their steady-state level. Finally, OGT silencing or OGA inhibition destabilizes MCM2/6 and MCM4/7 interactions in the chromatin-enriched fraction. In conclusion, OGT is a new partner of the MCM2-7 complex and O-GlcNAcylation homeostasis might regulate MCM2-7 complex by regulating the chromatin loading of MCM6 and MCM7 and stabilizing MCM/MCM interactions.
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Affiliation(s)
- Maïté Leturcq
- Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France
| | - Marlène Mortuaire
- Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France
| | - Stéphan Hardivillé
- Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France
| | - Céline Schulz
- Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France
| | - Tony Lefebvre
- Univ. Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59000, Lille, France
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11
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Abstract
Recently published structural and functional analyses of the CMG complex have provided insight into the mechanism of its DNA helicase function and into the distinct roles of its central six component proteins MCM2-MCM7 (MCM2-7). To activate CMG helicase, the two protein kinases CDK and DDK, as well as MCM10, are required. In addition to the initiation of DNA replication, MCM function must be regulated at the DNA replication steps of elongation and termination. Polyubiquitylation of MCM7 is involved in terminating MCM function. Reinitiation of DNA replication in a single cell cycle, which is prevented mainly by CDK, is understood at the molecular level. MCM2-7 gene expression is regulated during cellular aging and the cell cycle, and the expression depends on oxygen concentration. These regulatory processes have been described recently. Genomic structural alteration, which is an essential element in cancer progression, is mainly generated by disruptions of DNA replication fork structures. A point mutation in MCM4 that disturbs MCM2-7 function results in genomic instability, leading to the generation of cancer cells. In this review, I focus on the following points: 1) function of the MCM2-7 complex, 2) activation of MCM2-7 helicase, 3) regulation of MCM2-7 function, 4) MCM2-7 expression, and 5) the role of MCM mutation in cancer progression.
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12
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Abstract
Nucleosomes compact and organize genetic material on a structural level. However, they also alter local chromatin accessibility through changes in their position, through the incorporation of histone variants, and through a vast array of histone posttranslational modifications. The dynamic nature of chromatin requires histone chaperones to process, deposit, and evict histones in different tissues and at different times in the cell cycle. This review focuses on the molecular details of canonical and variant H3-H4 histone chaperone pathways that lead to histone deposition on DNA as they are currently understood. Emphasis is placed on the most established pathways beginning with the folding, posttranslational modification, and nuclear import of newly synthesized H3-H4 histones. Next, we review the deposition of replication-coupled H3.1-H4 in S-phase and replication-independent H3.3-H4 via alternative histone chaperone pathways. Highly specialized histone chaperones overseeing the deposition of histone variants are also briefly discussed.
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Affiliation(s)
- Prerna Grover
- Genetics & Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada;
| | - Jonathon S Asa
- Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5G 0A4, Canada
| | - Eric I Campos
- Genetics & Genome Biology Program, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada; .,Department of Molecular Genetics, The University of Toronto, Toronto, Ontario M5G 0A4, Canada
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13
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Evrin C, Maman JD, Diamante A, Pellegrini L, Labib K. Histone H2A-H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin. EMBO J 2018; 37:embj.201899021. [PMID: 30104407 PMCID: PMC6166128 DOI: 10.15252/embj.201899021] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2018] [Revised: 06/18/2018] [Accepted: 07/24/2018] [Indexed: 11/13/2022] Open
Abstract
The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re‐deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone‐binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating‐type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone‐binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin‐derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone‐binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.
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Affiliation(s)
- Cecile Evrin
- MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK
| | - Joseph D Maman
- Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Aurora Diamante
- Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Luca Pellegrini
- Department of Biochemistry, University of Cambridge, Cambridge, UK
| | - Karim Labib
- MRC Protein Phosphorylation and Ubiquitylation Unit, Sir James Black Centre, School of Life Sciences, University of Dundee, Dundee, UK
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14
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Serra-Cardona A, Zhang Z. Replication-Coupled Nucleosome Assembly in the Passage of Epigenetic Information and Cell Identity. Trends Biochem Sci 2017; 43:136-148. [PMID: 29292063 DOI: 10.1016/j.tibs.2017.12.003] [Citation(s) in RCA: 79] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2017] [Revised: 12/07/2017] [Accepted: 12/09/2017] [Indexed: 12/31/2022]
Abstract
During S phase, replicated DNA must be assembled into nucleosomes using both newly synthesized and parental histones in a process that is tightly coupled to DNA replication. This DNA replication-coupled process is regulated by multitude of histone chaperones as well as by histone-modifying enzymes. In recent years novel insights into nucleosome assembly of new H3-H4 tetramers have been gained through studies on the classical histone chaperone CAF-1 and the identification of novel factors involved in this process. Moreover, in vitro reconstitution of chromatin replication has shed light on nucleosome assembly of parental H3-H4, a process that remains elusive. Finally, recent studies have revealed that the replication-coupled nucleosome assembly is important for the determination and maintenance of cell fate in multicellular organisms.
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Affiliation(s)
- Albert Serra-Cardona
- Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA; Department of Pediatrics, Columbia University, New York, NY 10032, USA; Department of Genetics and Development, Columbia University, New York, NY 10032, USA
| | - Zhiguo Zhang
- Institute for Cancer Genetics, Columbia University, New York, NY 10032, USA; Department of Pediatrics, Columbia University, New York, NY 10032, USA; Department of Genetics and Development, Columbia University, New York, NY 10032, USA.
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15
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Prado F, Maya D. Regulation of Replication Fork Advance and Stability by Nucleosome Assembly. Genes (Basel) 2017; 8:genes8020049. [PMID: 28125036 PMCID: PMC5333038 DOI: 10.3390/genes8020049] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2016] [Revised: 01/04/2017] [Accepted: 01/16/2017] [Indexed: 12/13/2022] Open
Abstract
The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging.
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Affiliation(s)
- Felix Prado
- Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), Spanish National Research Council (CSIC), Seville 41092, Spain.
| | - Douglas Maya
- Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), Spanish National Research Council (CSIC), Seville 41092, Spain.
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16
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Kurat CF, Yeeles JTP, Patel H, Early A, Diffley JFX. Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates. Mol Cell 2017; 65:117-130. [PMID: 27989438 PMCID: PMC5222724 DOI: 10.1016/j.molcel.2016.11.016] [Citation(s) in RCA: 180] [Impact Index Per Article: 22.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2016] [Revised: 10/17/2016] [Accepted: 11/07/2016] [Indexed: 11/16/2022]
Abstract
The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo.
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Affiliation(s)
- Christoph F Kurat
- Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK
| | - Joseph T P Yeeles
- Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK
| | - Harshil Patel
- Lincoln's Inn Fields Laboratory, Francis Crick Institute, London NW1 1AT, UK
| | - Anne Early
- Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK
| | - John F X Diffley
- Clare Hall Laboratory, Francis Crick Institute, South Mimms, Hertfordshire EN6 3LD, UK.
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17
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Hammond CM, Strømme CB, Huang H, Patel DJ, Groth A. Histone chaperone networks shaping chromatin function. Nat Rev Mol Cell Biol 2017; 18:141-158. [PMID: 28053344 DOI: 10.1038/nrm.2016.159] [Citation(s) in RCA: 376] [Impact Index Per Article: 47.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
The association of histones with specific chaperone complexes is important for their folding, oligomerization, post-translational modification, nuclear import, stability, assembly and genomic localization. In this way, the chaperoning of soluble histones is a key determinant of histone availability and fate, which affects all chromosomal processes, including gene expression, chromosome segregation and genome replication and repair. Here, we review the distinct structural and functional properties of the expanding network of histone chaperones. We emphasize how chaperones cooperate in the histone chaperone network and via co-chaperone complexes to match histone supply with demand, thereby promoting proper nucleosome assembly and maintaining epigenetic information by recycling modified histones evicted from chromatin.
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Affiliation(s)
- Colin M Hammond
- Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK-2200, Denmark
| | - Caroline B Strømme
- Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK-2200, Denmark
| | - Hongda Huang
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
| | - Dinshaw J Patel
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
| | - Anja Groth
- Biotech Research and Innovation Centre (BRIC) and Centre for Epigenetics, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen DK-2200, Denmark
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18
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Juríková M, Danihel Ľ, Polák Š, Varga I. Ki67, PCNA, and MCM proteins: Markers of proliferation in the diagnosis of breast cancer. Acta Histochem 2016; 118:544-52. [PMID: 27246286 DOI: 10.1016/j.acthis.2016.05.002] [Citation(s) in RCA: 425] [Impact Index Per Article: 47.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2015] [Revised: 04/05/2016] [Accepted: 05/16/2016] [Indexed: 12/22/2022]
Abstract
The proliferative activity of tumour cells represents an important prognostic marker in the diagnosis of cancer. One of the methods for assessing the proliferative activity of cells is the immunohistochemical detection of cell cycle-specific antigens. For example, Ki67, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance (MCM) proteins are standard markers of proliferation that are commonly used to assess the growth fraction of a cell population. The function of Ki67, the widely used marker of proliferation, still remains unclear. In contrast, PCNA and MCM proteins have been identified as important participants of DNA replication. All three proteins only manifest their expression during the cell division of normal and neoplastic cells. Since the expression of these proliferative markers was confirmed in several malignant tumours, their prognostic and predictive values have been evaluated to determine their significance in the diagnosis of cancer. This review offers insight into the discovery of the abovementioned proteins, as well as their current molecular and biological importance. In addition, the functions and properties of all three proteins and their use as markers of proliferation in the diagnosis of breast cancer are described. This work also reveals new findings about the role of Ki67 during the mitotic phase of the cell cycle. Finally, information is provided about the advantages and disadvantages of using all three antigens in the diagnosis of cancer.
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Affiliation(s)
- Miroslava Juríková
- Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Špitálska 24, 813 72 Bratislava, Slovakia.
| | - Ľudovít Danihel
- Institute of Pathological Anatomy, Faculty of Medicine, Comenius University in Bratislava, Špitálska 24, 813 72 Bratislava, Slovakia
| | - Štefan Polák
- Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Špitálska 24, 813 72 Bratislava, Slovakia
| | - Ivan Varga
- Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Špitálska 24, 813 72 Bratislava, Slovakia
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19
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Wang H, Wang M, Yang N, Xu RM. Structure of the quaternary complex of histone H3-H4 heterodimer with chaperone ASF1 and the replicative helicase subunit MCM2. Protein Cell 2016; 6:693-7. [PMID: 26186914 PMCID: PMC4537477 DOI: 10.1007/s13238-015-0190-0] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022] Open
Affiliation(s)
- Hong Wang
- />National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
- />University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Mingzhu Wang
- />National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
| | - Na Yang
- />National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
- />University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Rui-Ming Xu
- />National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 China
- />University of Chinese Academy of Sciences, Beijing, 100049 China
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20
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Almouzni G, Cedar H. Maintenance of Epigenetic Information. Cold Spring Harb Perspect Biol 2016; 8:8/5/a019372. [PMID: 27141050 DOI: 10.1101/cshperspect.a019372] [Citation(s) in RCA: 108] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The genome is subject to a diverse array of epigenetic modifications from DNA methylation to histone posttranslational changes. Many of these marks are somatically stable through cell division. This article focuses on our knowledge of the mechanisms governing the inheritance of epigenetic marks, particularly, repressive ones, when the DNA and chromatin template are duplicated in S phase. This involves the action of histone chaperones, nucleosome-remodeling enzymes, histone and DNA methylation binding proteins, and chromatin-modifying enzymes. Last, the timing of DNA replication is discussed, including the question of whether this constitutes an epigenetic mark that facilitates the propagation of epigenetic marks.
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Affiliation(s)
- Geneviève Almouzni
- Department of Nuclear Dynamics and Genome Plasticity, Institut Curie, Section de recherche, 75231 Paris Cedex 05, France
| | - Howard Cedar
- Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University Medical School, Ein Kerem, Jerusalem, Israel 91120
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21
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Rizvi I, Choudhury NR, Tuteja N. Arabidopsis thaliana MCM3 single subunit of MCM2-7 complex functions as 3' to 5' DNA helicase. PROTOPLASMA 2016; 253:467-75. [PMID: 25944245 DOI: 10.1007/s00709-015-0825-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Accepted: 04/27/2015] [Indexed: 05/09/2023]
Abstract
Minichromosome maintenance 2-7 (MCM2-7) proteins are conserved eukaryotic replicative factors essential for the DNA replication at its initiation and elongation step, and act as a licensing factor. The MCM2-7 and MCM4/6/7subcomplex exhibit DNA helicase activity, and are therefore regarded as the replicative helicase. The MCM proteins have not been studied in detail in plant system. Here, we present the biochemical characterization of Arabidopsis thaliana MCM3 single subunit and show that it exhibits in vitro unwinding and ATPase activities. AtMCM3 shows a greater unwinding activity with 5' forked partial DNA duplex substrate as compared to 3' forked and non-forked substrates. ATP and magnesium ion are indispensable for its DNA helicase activity. Specifically, ATP and dATP are the preferred nucleotides for its unwinding activity. The directionality of the AtMCM3 has been determined to be in 3' to 5' direction. The oligomerization status of AtMCM3 single subunit protein indicates that it is present in different multimeric forms. The unraveling of the helicase activity of AtMCM3 will provide better insights into the plant DNA replication.
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Affiliation(s)
- Irum Rizvi
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Nirupam Roy Choudhury
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India
| | - Narendra Tuteja
- Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.
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22
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Gao J, Wang Q, Dong C, Chen S, Qi Y, Liu Y. Whole Exome Sequencing Identified MCM2 as a Novel Causative Gene for Autosomal Dominant Nonsyndromic Deafness in a Chinese Family. PLoS One 2015. [PMID: 26196677 PMCID: PMC4510057 DOI: 10.1371/journal.pone.0133522] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
We report the genetic analysis of autosomal dominant, nonsyndromic, progressive sensorineural hearing loss in a Chinese family. Using whole exome sequencing, we identified a missense variant (c.130C>T, p.R44C) in the MCM2 gene, which has a pro-apoptosis effect and is involved in the initiation of eukaryotic genome replication. This missense variant is very likely to be the disease causing variant. It segregated with hearing loss in this pedigree, and was not found in the dbSNP database or databases of genomes and SNP in the Chinese population, in 76 patients with sporadic hearing loss, or in 145 normal individuals. We performed western blot and immunofluorescence to test the MCM2 protein expression in the cochlea of rats and guinea pigs, demonstrating that MCM2 was widely expressed in the cochlea and was also surprisingly expressed in the cytoplasm of terminally differentiated hair cells. We then transiently expressed the variant MCM2 cDNA in HEK293 cells, and found that these cells displayed a slight increase in apoptosis without any changes in proliferation or cell cycle, supporting the view that this variant is pathogenic. In summary, we have identified MCM2 as a novel gene responsible for nonsyndromic hearing loss of autosomal dominant inheritance in a Chinese family.
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Affiliation(s)
- Juanjuan Gao
- Department of Otolaryngology, Head and Neck Surgery, Peking University First Hospital, Beijing, China
| | - Qi Wang
- Department of Otolaryngology, Head and Neck Surgery, Peking University First Hospital, Beijing, China
| | - Cheng Dong
- Department of Otolaryngology, Head and Neck Surgery, Peking University First Hospital, Beijing, China
| | - Siqi Chen
- Department of Otolaryngology, Head and Neck Surgery, Peking University First Hospital, Beijing, China
| | - Yu Qi
- Department of central laboratory, Peking University First Hospital, Beijing, China
| | - Yuhe Liu
- Department of Otolaryngology, Head and Neck Surgery, Peking University First Hospital, Beijing, China
- * E-mail:
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23
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Huang H, Strømme CB, Saredi G, Hödl M, Strandsby A, González-Aguilera C, Chen S, Groth A, Patel DJ. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks. Nat Struct Mol Biol 2015; 22:618-26. [PMID: 26167883 DOI: 10.1038/nsmb.3055] [Citation(s) in RCA: 180] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 06/04/2015] [Indexed: 12/11/2022]
Abstract
During DNA replication, chromatin is reassembled by recycling of modified old histones and deposition of new ones. How histone dynamics integrates with DNA replication to maintain genome and epigenome information remains unclear. Here, we reveal how human MCM2, part of the replicative helicase, chaperones histones H3-H4. Our first structure shows an H3-H4 tetramer bound by two MCM2 histone-binding domains (HBDs), which hijack interaction sites used by nucleosomal DNA. Our second structure reveals MCM2 and ASF1 cochaperoning an H3-H4 dimer. Mutational analyses show that the MCM2 HBD is required for MCM2-7 histone-chaperone function and normal cell proliferation. Further, we show that MCM2 can chaperone both new and old canonical histones H3-H4 as well as H3.3 and CENPA variants. The unique histone-binding mode of MCM2 thus endows the replicative helicase with ideal properties for recycling histones genome wide during DNA replication.
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Affiliation(s)
- Hongda Huang
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
| | - Caroline B Strømme
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Giulia Saredi
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Martina Hödl
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Anne Strandsby
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Cristina González-Aguilera
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Shoudeng Chen
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
| | - Anja Groth
- Biotech Research and Innovation Centre (BRIC) Centre for Epigenetics, University of Copenhagen, Copenhagen, Denmark
| | - Dinshaw J Patel
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA
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24
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Annunziato AT. The Fork in the Road: Histone Partitioning During DNA Replication. Genes (Basel) 2015; 6:353-71. [PMID: 26110314 PMCID: PMC4488668 DOI: 10.3390/genes6020353] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2015] [Revised: 06/15/2015] [Accepted: 06/16/2015] [Indexed: 12/22/2022] Open
Abstract
In the following discussion the distribution of histones at the replication fork is examined, with specific attention paid to the question of H3/H4 tetramer "splitting." After a presentation of early experiments surrounding this topic, more recent contributions are detailed. The implications of these findings with respect to the transmission of histone modifications and epigenetic models are also addressed.
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Affiliation(s)
- Anthony T Annunziato
- Biology Department, Boston College, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA.
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25
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Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate. Mol Cell Biol 2015; 35:2131-43. [PMID: 25870112 DOI: 10.1128/mcb.01357-14] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2014] [Accepted: 04/05/2015] [Indexed: 01/01/2023] Open
Abstract
The DNA replication checkpoint (DRC) monitors and responds to stalled replication forks to prevent genomic instability. How core replication factors integrate into this phosphorylation cascade is incompletely understood. Here, through analysis of a unique mcm allele targeting a specific ATPase active site (mcm2DENQ), we show that the Mcm2-7 replicative helicase has a novel DRC function as part of the signal transduction cascade. This allele exhibits normal downstream mediator (Mrc1) phosphorylation, implying DRC sensor kinase activation. However, the mutant also exhibits defective effector kinase (Rad53) activation and classic DRC phenotypes. Our previous in vitro analysis showed that the mcm2DENQ mutation prevents a specific conformational change in the Mcm2-7 hexamer. We infer that this conformational change is required for its DRC role and propose that it allosterically facilitates Rad53 activation to ensure a replication-specific checkpoint response.
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26
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Hesketh EL, Parker-Manuel RP, Chaban Y, Satti R, Coverley D, Orlova EV, Chong JPJ. DNA induces conformational changes in a recombinant human minichromosome maintenance complex. J Biol Chem 2015; 290:7973-9. [PMID: 25648893 PMCID: PMC4367295 DOI: 10.1074/jbc.m114.622738] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2014] [Revised: 02/02/2015] [Indexed: 11/30/2022] Open
Abstract
ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.
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Affiliation(s)
- Emma L Hesketh
- From the Department of Biology, University of York, York YO10 5DD and
| | | | - Yuriy Chaban
- the Department of Crystallography, Birkbeck College London, London WC1E 7HX, United Kingdom
| | - Rabab Satti
- From the Department of Biology, University of York, York YO10 5DD and
| | - Dawn Coverley
- From the Department of Biology, University of York, York YO10 5DD and
| | - Elena V Orlova
- the Department of Crystallography, Birkbeck College London, London WC1E 7HX, United Kingdom
| | - James P J Chong
- From the Department of Biology, University of York, York YO10 5DD and
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27
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Richet N, Liu D, Legrand P, Velours C, Corpet A, Gaubert A, Bakail M, Moal-Raisin G, Guerois R, Compper C, Besle A, Guichard B, Almouzni G, Ochsenbein F. Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork. Nucleic Acids Res 2015; 43:1905-17. [PMID: 25618846 PMCID: PMC4330383 DOI: 10.1093/nar/gkv021] [Citation(s) in RCA: 102] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.
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Affiliation(s)
- Nicolas Richet
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Danni Liu
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | | | - Christophe Velours
- Laboratoire d'Enzymologie et de Biologie Structurale, CNRS UPR 3082, 1 avenue de la Terrasse, Gif-sur-Yvette, F-91190, France
| | - Armelle Corpet
- Institut Curie, Centre de Recherche, CNRS UMR 3664, Equipe Labellisée Ligue contre le Cancer, and Université Pierre et Marie Curie, Université Sorbonne PSL*, Paris, F-75248, France
| | - Albane Gaubert
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - May Bakail
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Gwenaelle Moal-Raisin
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Raphael Guerois
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Christel Compper
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Arthur Besle
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Berengère Guichard
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
| | - Genevieve Almouzni
- Institut Curie, Centre de Recherche, CNRS UMR 3664, Equipe Labellisée Ligue contre le Cancer, and Université Pierre et Marie Curie, Université Sorbonne PSL*, Paris, F-75248, France
| | - Françoise Ochsenbein
- CEA, iBiTec-S, SB2SM, Laboratoire de Biologie Structurale et Radiobiologie, France Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Batiment 144, Gif-sur-Yvette, F-91191, France
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28
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Foltman M, Evrin C, De Piccoli G, Jones RC, Edmondson RD, Katou Y, Nakato R, Shirahige K, Labib K. Eukaryotic replisome components cooperate to process histones during chromosome replication. Cell Rep 2013; 3:892-904. [PMID: 23499444 DOI: 10.1016/j.celrep.2013.02.028] [Citation(s) in RCA: 151] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2012] [Revised: 12/30/2012] [Accepted: 02/28/2013] [Indexed: 11/30/2022] Open
Abstract
DNA unwinding at eukaryotic replication forks displaces parental histones, which must be redeposited onto nascent DNA in order to preserve chromatin structure. By screening systematically for replisome components that pick up histones released from chromatin into a yeast cell extract, we found that the Mcm2 helicase subunit binds histones cooperatively with the FACT (facilitiates chromatin transcription) complex, which helps to re-establish chromatin during transcription. FACT does not associate with the Mcm2-7 helicase at replication origins during G1 phase but is subsequently incorporated into the replisome progression complex independently of histone binding and uniquely among histone chaperones. The amino terminal tail of Mcm2 binds histones via a conserved motif that is dispensable for DNA synthesis per se but helps preserve subtelomeric chromatin, retain the 2 micron minichromosome, and support growth in the absence of Ctf18-RFC. Our data indicate that the eukaryotic replication and transcription machineries use analogous assemblies of multiple chaperones to preserve chromatin integrity.
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Affiliation(s)
- Magdalena Foltman
- Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK
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29
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Uno S, You Z, Masai H. Purification of replication factors using insect and mammalian cell expression systems. Methods 2012; 57:214-21. [PMID: 22800621 DOI: 10.1016/j.ymeth.2012.06.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2012] [Revised: 06/12/2012] [Accepted: 06/25/2012] [Indexed: 10/28/2022] Open
Abstract
Purification of factors for DNA replication in an amount sufficient for detailed biochemical characterization is essential to elucidating its mechanisms. Insect cell expression systems are commonly used for purification of the factors proven to be difficult to deal with in bacteria. We describe first the detailed protocols for purification of mammalian Mcm complexes including the Mcm2/3/4/5/6/7 heterohexamer expressed in insect cells. We then describe a convenient and economical system in which large-sized proteins and multi-factor complexes can be transiently overexpressed in human 293T cells and be rapidly purified in a large quantity. We describe various expression vectors and detailed methods for transfection and purification of various replication factors which have been difficult to obtain in a sufficient amount in other systems. Availability of efficient methods to overproduce and purify the proteins that have been challenging would facilitate the enzymatic analyses of the processes of DNA replication.
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Affiliation(s)
- Shuji Uno
- Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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30
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Abe S, Kurata M, Suzuki S, Yamamoto K, Aisaki KI, Kanno J, Kitagawa M. Minichromosome maintenance 2 bound with retroviral Gp70 is localized to cytoplasm and enhances DNA-damage-induced apoptosis. PLoS One 2012; 7:e40129. [PMID: 22768239 PMCID: PMC3387003 DOI: 10.1371/journal.pone.0040129] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Accepted: 06/01/2012] [Indexed: 11/19/2022] Open
Abstract
The interaction of viral proteins with host-cellular proteins elicits the activation of cellular signal transduction pathways and possibly leads to viral pathogenesis as well as cellular biological events. Apoptotic signals induced by DNA-damage are remarkably up-regulated by Friend leukemia virus (FLV) exclusively in C3H hosts; however, the mechanisms underlying the apoptosis enhancement and host-specificity are unknown. Here, we show that C3H mouse-derived hematopoietic cells originally express higher levels of the minichromosome maintenance (MCM) 2 protein than BALB/c- or C57BL/6-deriverd cells, and undergo more frequent apoptosis following doxorubicin-induced DNA-damage in the presence of the FLV envelope protein gp70. Dual transfection with gp70/Mcm2 reproduced doxorubicin-induced apoptosis even in BALB/c-derived 3T3 cells. Immunoprecipitation assays using various deletion mutants of MCM2 revealed that gp70 bound to the nuclear localization signal (NLS) 1 (amino acids 18–24) of MCM2, interfered with the function of NLS2 (amino acids 132–152), and suppressed the normal nuclear-import of MCM2. Cytoplasmic MCM2 reduced the activity of protein phosphatase 2A (PP2A) leading to the subsequent hyperphosphorylation of DNA-dependent protein kinase (DNA-PK). Phosphorylated DNA-PK exhibited elevated kinase activity to phosphorylate P53, thereby up-regulating p53-dependent apoptosis. An apoptosis-enhancing domain was identified in the C-terminal portion (amino acids 703–904) of MCM2. Furthermore, simultaneous treatment with FLV and doxorubicin extended the survival of SCID mice bearing 8047 leukemia cells expressing high levels of MCM2. Thus, depending on its subcellular localization, MCM2 plays different roles. It participates in DNA replication in the nucleus as shown previously, and enhances apoptosis in the cytoplasm.
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Affiliation(s)
- Shinya Abe
- Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Morito Kurata
- Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Shiho Suzuki
- Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Kouhei Yamamoto
- Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
| | - Ken-ichi Aisaki
- Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan
| | - Jun Kanno
- Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan
| | - Masanobu Kitagawa
- Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
- * E-mail:
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31
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Watanabe E, Ohara R, Ishimi Y. Effect of an MCM4 mutation that causes tumours in mouse on human MCM4/6/7 complex formation. J Biochem 2012; 152:191-8. [PMID: 22668557 DOI: 10.1093/jb/mvs060] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
It has been reported that a point mutation of minichromosome maintenance (MCM)4 causes mammary carcinoma, and it deregulates DNA replication to produce abnormal chromosome structures. To understand the effect of this mutation at level of MCM2-7 interaction, we examined the effect of the same mutation of human MCM4 on the complex formation with MCM6 and MCM7 in insect cells. Human MCM4/6/7 complexes containing the mutated MCM4 were formed, but the hexameric complex formation was not evident in comparison with those containing wild-type MCM4. In binary expression of MCM4 and MCM6, decreased levels of MCM6 were recovered with the mutated MCM4, compared with wild-type MCM4. These results suggest that this mutation of MCM4 perturbs proper interaction with MCM6 to affect complex formation of MCM4/6/7 that is a core structure of MCM2-7 complex. Consistent with this notion, nuclear localization and MCM complex formation of forcedly expressed MCM4 in human cells are affected by this mutation. Thus, the defect of this mutant MCM4 in interacting with MCM6 may generate a decreased level of chromatin binding of MCM2-7 complex.
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Affiliation(s)
- Emi Watanabe
- College of Science, Ibaraki University, 2-1-1 Bunkyo, Mito, Ibaraki 351-8511, Japan
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32
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Ishikawa K, Ohsumi T, Tada S, Natsume R, Kundu LR, Nozaki N, Senda T, Enomoto T, Horikoshi M, Seki M. Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication. Genes Cells 2011; 16:1050-62. [DOI: 10.1111/j.1365-2443.2011.01549.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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33
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The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function. BMC BIOCHEMISTRY 2010; 11:37. [PMID: 20860810 PMCID: PMC2949612 DOI: 10.1186/1471-2091-11-37] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/30/2010] [Accepted: 09/22/2010] [Indexed: 12/29/2022]
Abstract
BACKGROUND Minichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7. RESULTS Here, we show that Saccharomyces cerevisiae Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer. CONCLUSIONS The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.
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34
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Sakamoto M, Noguchi S, Kawashima S, Okada Y, Enomoto T, Seki M, Horikoshi M. Global analysis of mutual interaction surfaces of nucleosomes with comprehensive point mutants. Genes Cells 2010; 14:1271-330. [PMID: 19903202 DOI: 10.1111/j.1365-2443.2009.01350.x] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
The surfaces of core histones in nucleosome are exposed as required for factor recognition, or buried for histone-DNA and histone-histone interactions. To understand the mechanisms by which nucleosome structure and function are coordinately altered in DNA-mediated reactions, it is essential to define the roles of both exposed and buried residues and their functional relationships. For this purpose, we developed GLASP (GLobal Analysis of Surfaces by Point mutation) and GLAMP (GLobal Analysis of Mutual interaction surfaces of multi-subunit protein complex by Point mutation) strategies, both of which are comprehensive analyses by point mutagenesis of exposed and buried residues in nucleosome, respectively. Four distinct DNA-mediated reactions evaluated by Ty suppression (the Spt(-) phenotype), and sensitivities to 6-azauracil (6AU), hydroxyurea (HU), and methyl methanesulfonate (MMS), require common and different GLAMP residues. Mutated GLAMP residues at the interface between histones H2A and H2B mainly affect the Spt(-) phenotype but not HU and MMS sensitivities. Interestingly, among the mutated GLAMP residues surrounding the histone H3-H3' interface, some equally affect the Spt(-) phenotype, and HU and MMS sensitivities, whereas others differentially affect the Spt(-) phenotype, and HU and MMS sensitivities. Based on these and other results, the functional relationships among chromatin factors and GLASP and GLAMP residues provide insights into nucleosome disassembly/assembly processes in DNA-mediated reactions.
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Affiliation(s)
- Makoto Sakamoto
- Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan
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35
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Stead BE, Sorbara CD, Brandl CJ, Davey MJ. ATP binding and hydrolysis by Mcm2 regulate DNA binding by Mcm complexes. J Mol Biol 2009; 391:301-13. [PMID: 19540846 DOI: 10.1016/j.jmb.2009.06.038] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2009] [Revised: 06/10/2009] [Accepted: 06/16/2009] [Indexed: 01/20/2023]
Abstract
The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2(KA)-7. Moreover, together with the finding that Mcm2(K549A) does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes.
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Affiliation(s)
- Brent E Stead
- Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, Ontario, Canada
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36
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Geha S, Pallud J, Junier MP, Devaux B, Leonard N, Chassoux F, Chneiweiss H, Daumas-Duport C, Varlet P. NG2+/Olig2+ cells are the major cycle-related cell population of the adult human normal brain. Brain Pathol 2009; 20:399-411. [PMID: 19486010 DOI: 10.1111/j.1750-3639.2009.00295.x] [Citation(s) in RCA: 112] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
A persistent cycling cell population in the normal adult human brain is well established. Neural stem cells or neural progenitors have been identified in the subventricular zone and the dentate gyrus subgranular layer (SGL), two areas of persistent neurogenesis. Cycling cells in other human normal brain areas, however, remains to be established. Here, we determined the distribution and identity of these cells in the cortex, the white matter and the hippocampal formation of adult patients with and without chronic temporal lobe epilepsy using immunohistochemistry for the cell cycle markers Ki-67 (Mib-1) and minichromosome maintenance protein 2. Rare proliferative neuronal precursors expressing the neuronal antigen neuronal nuclei were restricted to the SGL. In contrast, the oligodendrocyte progenitor cell markers Olig2 and the surface antigen NG2 were expressed by the vast majority of cycling cells scattered throughout the cortex and white matter of both control and epileptic patients. Most of these cycling cells were in early G1 phase, and were significantly more numerous in epileptic than in non-epileptic patients. These results provide evidence for a persistent gliogenesis in the human cortex and white matter that is enhanced in an epileptic environment.
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Affiliation(s)
- Sameh Geha
- Department of Neuropathology, Sainte-Anne Hospital, Paris, France
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37
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Ishimi Y, Sugiyama T, Nakaya R, Kanamori M, Kohno T, Enomoto T, Chino M. Effect of heliquinomycin on the activity of human minichromosome maintenance 4/6/7 helicase. FEBS J 2009; 276:3382-91. [PMID: 19438708 DOI: 10.1111/j.1742-4658.2009.07064.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half-maximal inhibitory concentration (IC(50)) of 1.4-4 microM, was found to inhibit the DNA helicase activity of the human minichromosome maintenance (MCM) 4/6/7 complex at an IC(50) value of 2.4 microM. In contrast, 14 microM heliquinomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A. At IC(50) values of 25 and 6.5 microM, heliquinomycin inhibited the RNA priming and DNA polymerization activities, respectively, of human DNA polymerase-alpha/primase. Thus, of the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliquinomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo. It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence of single-stranded DNA. In contrast, heliquinomycin at an IC(50) value of 5.2 microM inhibited the ATPase activity of the MCM4/6/7 complex in the presence of single-stranded DNA. This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA.
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38
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Groth A. Replicating chromatin: a tale of histonesThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB’s 51st Annual Meeting – Epigenetics and Chromatin Dynamics, and has undergone the Journal’s usual peer review process. Biochem Cell Biol 2009; 87:51-63. [DOI: 10.1139/o08-102] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Chromatin serves structural and functional roles crucial for genome stability and correct gene expression. This organization must be reproduced on daughter strands during replication to maintain proper overlay of epigenetic fabric onto genetic sequence. Nucleosomes constitute the structural framework of chromatin and carry information to specify higher-order organization and gene expression. When replication forks traverse the chromosomes, nucleosomes are transiently disrupted, allowing the replication machinery to gain access to DNA. Histone recycling, together with new deposition, ensures reassembly on nascent DNA strands. The aim of this review is to discuss how histones — new and old — are handled at the replication fork, highlighting new mechanistic insights and revisiting old paradigms.
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Affiliation(s)
- Anja Groth
- Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen, Denmark (e-mail: )
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39
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Cho JH, Kim HB, Kim HS, Choi SB. Identification and characterization of a rice MCM2 homologue required for DNA replication. BMB Rep 2008; 41:581-6. [PMID: 18755073 DOI: 10.5483/bmbrep.2008.41.8.581] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The pre-replication complex (pre-RC), including the core hexameric MCM2-7 complex, ensures that the eukaryotic genome is replicated only once per cell division cycle. In this study, we identified a rice minichromosome maintenance (MCM) homologue (OsMCM2) that functionally complemented fission yeast MCM2 (CDC19) mutants. We found OsMCM2 transcript expression in roots, leaves, and seeds, although expression levels differed slightly among the organs. Likewise, the OsMCM2 protein was ubiquitously expressed, but it was downregulated when nutritients were limiting, indicating that MCM2 expression (and therefore cell cycle progression) requires adequate nutrition. Yeast two-hybrid and GST pull-down assays demonstrated that OsMCM2 interacted with the COP9 signalosome 5 (CSN5). Taken as a whole, our results indicated that OsMCM2 functions as a subunit of the rice MCM complex and interacts with CSN5 during developmental regulation.
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Affiliation(s)
- Jae Han Cho
- Department of Biological Sciences, Myongji University, Yongin, Korea
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40
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Kanter DM, Bruck I, Kaplan DL. Mcm subunits can assemble into two different active unwinding complexes. J Biol Chem 2008; 283:31172-82. [PMID: 18801730 DOI: 10.1074/jbc.m804686200] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
The replication fork helicase in eukaryotes is a large complex that is composed of Mcm2-7, Cdc45, and GINS. The Mcm2-7 proteins form a heterohexameric ring that hydrolyzes ATP and provide the motor function for this unwinding complex. A comprehensive study of how individual Mcm subunit biochemical activities relate to unwinding function has not been accomplished. We studied the mechanism of the Mcm4-Mcm6-Mcm7 complex, a useful model system because this complex has helicase activity in vitro. We separately purified each of three Mcm subunits until they were each nuclease-free, and we then examined the biochemical properties of different combinations of Mcm subunits. We found that Mcm4 and Mcm7 form an active unwinding assembly. The addition of Mcm6 to Mcm4/Mcm7 results in the formation of an active Mcm4/Mcm6/Mcm7 helicase assembly. The Mcm4-Mcm7 complex forms a ringed-shaped hexamer that unwinds DNA with 3' to 5' polarity by a steric exclusion mechanism, similar to Mcm4/Mcm6/Mcm7. The Mcm4-Mcm7 complex has a high level of ATPase activity that is further stimulated by DNA. The ability of different Mcm mixtures to form rings or exhibit DNA stimulation of ATPase activity correlates with the ability of these complexes to unwind DNA. The Mcm4/Mcm7 and Mcm4/Mcm6/Mcm7 assemblies can open to load onto circular DNA to initiate unwinding. We conclude that the Mcm subunits are surprisingly flexible and dynamic in their ability to interact with one another to form active unwinding complexes.
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Affiliation(s)
- Diane M Kanter
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA
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41
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Functional conservation of beta-hairpin DNA binding domains in the Mcm protein of Methanobacterium thermoautotrophicum and the Mcm5 protein of Saccharomyces cerevisiae. Genetics 2008; 179:1757-68. [PMID: 18660534 DOI: 10.1534/genetics.108.088690] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Mcm proteins are an important family of evolutionarily conserved helicases required for DNA replication in eukaryotes. The eukaryotic Mcm complex consists of six paralogs that form a heterohexameric ring. Because the intact Mcm2-7 hexamer is inactive in vitro, it has been difficult to determine the precise function of the different subunits. The solved atomic structure of an archaeal minichromosome maintenance (MCM) homolog provides insight into the function of eukaryotic Mcm proteins. The N-terminal positively charged central channel in the archaeal molecule consists of beta-hairpin domains essential for DNA binding in vitro. Eukaryotic Mcm proteins also have beta-hairpin domains, but their function is unknown. With the archaeal atomic structure as a guide, yeast molecular genetics was used to query the function of the beta-hairpin domains in vivo. A yeast mcm5 mutant with beta-hairpin mutations displays defects in the G1/S transition of the cell cycle, the initiation phase of DNA replication, and in the binding of the entire Mcm2-7 complex to replication origins. A similar mcm4 mutation is synthetically lethal with the mcm5 mutation. Therefore, in addition to its known regulatory role, Mcm5 protein has a positive role in origin binding, which requires coordination by all six Mcm2-7 subunits in the hexamer.
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42
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Kakusho N, Taniyama C, Masai H. Identification of Stimulators and Inhibitors of Cdc7 Kinase in Vitro. J Biol Chem 2008; 283:19211-8. [DOI: 10.1074/jbc.m803113200] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
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43
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Komamura-Kohno Y, Tanaka R, Omori A, Kohno T, Ishimi Y. Biochemical characterization of fragmented human MCM2. FEBS J 2008; 275:727-38. [PMID: 18190532 DOI: 10.1111/j.1742-4658.2007.06239.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The molecular dissection of human MCM2, a constituent of MCM2-7 licensing factor complex, was performed to identify the region responsible for its biochemical activities. Partial digestion with trypsin dissected the MCM2 protein into a central region (148-676) containing ATPase motifs and a C-terminal region (677-895). These two fragments, along with three other fragments (148-441, 442-676 and 442-895), were produced using the wheat germ cell-free system and were examined for their ability to inhibit MCM4/6/7 helicase activity. Two fragments (442-895 and 677-895) containing the C-terminus were partly inhibitory to the activity. Further dissection revealed that one fragment (713-895) has strong inhibitory activity. The inhibitory activity of the smaller fragments derived from the C-terminal region correlated with their ability to inhibit SV40 T antigen helicase activity and also with their ability to bind to ssDNA, which has been shown by gel mobility shift analysis. These results strongly suggest that the MCM2 fragments derived from the C-terminal region inhibit DNA helicase activity through their ability to bind to ssDNA. In contrast, two fragments (148-441 and 442-676) from the central region were mainly responsible for the interaction between MCM2 and MCM4, and this was revealed by a pulldown analysis using MCM4 protein beads. Finally, only complete MCM2, not the smaller fragments, could disassemble the MCM4/6/7 hexamer into the MCM2/4/6/7 tetramer.
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Affiliation(s)
- Yuki Komamura-Kohno
- Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Tokyo, Japan
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44
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Martin L. The Replicon Initiation Burst Released by Reoxygenation of Hypoxic T24 Cells is Accompanied by Changes of MCM2 and Cdc7. BMB Rep 2007; 40:805-13. [DOI: 10.5483/bmbrep.2007.40.5.805] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
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45
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Moyer SE, Lewis PW, Botchan MR. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proc Natl Acad Sci U S A 2006; 103:10236-10241. [PMID: 16798881 PMCID: PMC1482467 DOI: 10.1073/pnas.0602400103] [Citation(s) in RCA: 546] [Impact Index Per Article: 28.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The protein Cdc45 plays a critical but poorly understood role in the initiation and elongation stages of eukaryotic DNA replication. To study Cdc45's function in DNA replication, we purified Cdc45 protein from Drosophila embryo extracts by a combination of traditional and immunoaffinity chromatography steps and found that the protein exists in a stable, high-molecular-weight complex with the Mcm2-7 hexamer and the GINS tetramer. The purified Cdc45/Mcm2-7/GINS complex is associated with an active ATP-dependent DNA helicase function. RNA interference knock-down experiments targeting the GINS and Cdc45 components establish that the proteins are required for the S phase transition in Drosophila cells. The data suggest that this complex forms the core helicase machinery for eukaryotic DNA replication.
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Affiliation(s)
- Stephen E Moyer
- Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720
| | - Peter W Lewis
- Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720
| | - Michael R Botchan
- Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720
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Komamura-Kohno Y, Karasawa-Shimizu K, Saitoh T, Sato M, Hanaoka F, Tanaka S, Ishimi Y. Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells. FEBS J 2006; 273:1224-39. [PMID: 16519687 DOI: 10.1111/j.1742-4658.2006.05146.x] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
MCM4, a subunit of a putative replicative helicase, is phosphorylated during the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be performed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phosphorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phosphorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2/cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not generally colocalized with replicating DNA. Unexpectedly, MCM4 phosphorylated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle.
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Pacek M, Tutter AV, Kubota Y, Takisawa H, Walter JC. Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Mol Cell 2006; 21:581-7. [PMID: 16483939 DOI: 10.1016/j.molcel.2006.01.030] [Citation(s) in RCA: 291] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2005] [Revised: 01/20/2006] [Accepted: 01/26/2006] [Indexed: 10/25/2022]
Abstract
Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.
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Affiliation(s)
- Marcin Pacek
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
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Ajioka I, Maeda T, Nakajima K. Identification of ventricular-side-enriched molecules regulated in a stage-dependent manner during cerebral cortical development. Eur J Neurosci 2006; 23:296-308. [PMID: 16420439 DOI: 10.1111/j.1460-9568.2005.04544.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
Radial glial cells are the main component of the embryonic cortical ventricular zone (VZ), producing deep-layer excitatory neurons in the early stage and upper-layer excitatory neurons in the late stage of development. Previous studies have suggested that the laminar fate of deep-layer neurons might be determined by early-stage-specific secretory or transmembrane molecules (S/TMs) in the VZ. However, the different properties required to produce the different types of neurons in early-stage and late-stage VZ cells are largely unknown. Herein, we investigated the stage-dependent transcriptional profiles of the ventricular side of the mouse cortex, which was manually dissected at embryonic day (E)12, E14 and E16, and identified 3985 'VZ-enriched' genes, regulated stage-dependently, by GeneChip analysis. These molecules were classified into nine types based on stage-dependent regulation patterns. Prediction programs for the S/TMs revealed 659 'VZ-enriched' S/TMs. In situ hybridization and real-time PCR analysis for several of these molecules showed results consistent with the statistical analysis of the GeneChip experiments. Moreover, we identified 17 cell cycle-related early-stage and 'VZ-enriched' molecules. These molecules included not only those involved in cell cycle progression, but also essential molecules for DNA double-strand break repair, such as Rad51 and Rpa1. These results suggest that the early stage-VZ cells, which produce both deep- and upper-layer neurons, and the late-stage VZ cells, which produce only upper-layer neurons, are intrinsically different. The gene lists presented here will be useful for the investigation of stage-dependent changes in VZ cells and their regulatory mechanisms in the developing cortex.
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Affiliation(s)
- Itsuki Ajioka
- Department of Anatomy, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
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Zhu W, Abbas T, Dutta A. DNA replication and genomic instability. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2006; 570:249-79. [PMID: 18727504 DOI: 10.1007/1-4020-3764-3_9] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Affiliation(s)
- Wenge Zhu
- Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA
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Liku ME, Nguyen VQ, Rosales AW, Irie K, Li JJ. CDK phosphorylation of a novel NLS-NES module distributed between two subunits of the Mcm2-7 complex prevents chromosomal rereplication. Mol Biol Cell 2005; 16:5026-39. [PMID: 16093348 PMCID: PMC1237101 DOI: 10.1091/mbc.e05-05-0412] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2005] [Revised: 07/26/2005] [Accepted: 08/02/2005] [Indexed: 11/11/2022] Open
Abstract
Cyclin-dependent kinases (CDKs) use multiple mechanisms to block reassembly of prereplicative complexes (pre-RCs) at replication origins to prevent inappropriate rereplication. In Saccharomyces cerevisiae, one of these mechanisms promotes the net nuclear export of a pre-RC component, the Mcm2-7 complex, during S, G2, and M phases. Here we identify two partial nuclear localization signals (NLSs) on Mcm2 and Mcm3 that are each necessary, but not sufficient, for nuclear localization of the Mcm2-7 complex. When brought together in cis, however, the two partial signals constitute a potent NLS, sufficient for robust nuclear localization when fused to an otherwise cytoplasmic protein. We also identify a Crm1-dependent nuclear export signal (NES) adjacent to the Mcm3 NLS. Remarkably, the Mcm2-Mcm3 NLS and the Mcm3 NES are sufficient to form a transport module that recapitulates the cell cycle-regulated localization of the entire Mcm2-7 complex. Moreover, we show that CDK regulation promotes net export by phosphorylation of the Mcm3 portion of this module and that nuclear export of the Mcm2-7 complex is sufficient to disrupt replication initiation. We speculate that the distribution of partial transport signals among distinct subunits of a complex may enhance the specificity of protein localization and raises the possibility that previously undetected distributed transport signals are used by other multiprotein complexes.
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Affiliation(s)
- Muluye E Liku
- Department of Biochemistry, University of California, San Francisco, CA 94143-2200, USA
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