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Kingsley G, Skagia A, Passaretti P, Fernandez-Cuesta C, Reynolds-Winczura A, Koscielniak K, Gambus A. DONSON facilitates Cdc45 and GINS chromatin association and is essential for DNA replication initiation. Nucleic Acids Res 2023; 51:9748-9763. [PMID: 37638758 PMCID: PMC10570026 DOI: 10.1093/nar/gkad694] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 08/02/2023] [Accepted: 08/17/2023] [Indexed: 08/29/2023] Open
Abstract
Faithful cell division is the basis for the propagation of life and DNA replication must be precisely regulated. DNA replication stress is a prominent endogenous source of genome instability that not only leads to ageing, but also neuropathology and cancer development in humans. Specifically, the issues of how vertebrate cells select and activate origins of replication are of importance as, for example, insufficient origin firing leads to genomic instability and mutations in replication initiation factors lead to the rare human disease Meier-Gorlin syndrome. The mechanism of origin activation has been well characterised and reconstituted in yeast, however, an equal understanding of this process in higher eukaryotes is lacking. The firing of replication origins is driven by S-phase kinases (CDKs and DDK) and results in the activation of the replicative helicase and generation of two bi-directional replication forks. Our data, generated from cell-free Xenopus laevis egg extracts, show that DONSON is required for assembly of the active replicative helicase (CMG complex) at origins during replication initiation. DONSON has previously been shown to be essential during DNA replication, both in human cells and in Drosophila, but the mechanism of DONSON's action was unknown. Here we show that DONSON's presence is essential for replication initiation as it is required for Cdc45 and GINS association with Mcm2-7 complexes and helicase activation. To fulfil this role, DONSON interacts with the initiation factor, TopBP1, in a CDK-dependent manner. Following its initiation role, DONSON also forms a part of the replisome during the elongation stage of DNA replication. Mutations in DONSON have recently been shown to lead to the Meier-Gorlin syndrome; this novel replication initiation role of DONSON therefore provides the explanation for the phenotypes caused by DONSON mutations in patients.
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Affiliation(s)
- Georgia Kingsley
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Aggeliki Skagia
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Paolo Passaretti
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Cyntia Fernandez-Cuesta
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Alicja Reynolds-Winczura
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Kinga Koscielniak
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
| | - Agnieszka Gambus
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, UK
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Willemsen M, Barber JS, Nieuwenhove EV, Staels F, Gerbaux M, Neumann J, Prezzemolo T, Pasciuto E, Lagou V, Boeckx N, Filtjens J, De Visscher A, Matthys P, Schrijvers R, Tousseyn T, O'Driscoll M, Bucciol G, Schlenner S, Meyts I, Humblet-Baron S, Liston A. Homozygous DBF4 mutation as a cause of severe congenital neutropenia. J Allergy Clin Immunol 2023; 152:266-277. [PMID: 36841265 DOI: 10.1016/j.jaci.2023.02.016] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Revised: 01/23/2023] [Accepted: 02/16/2023] [Indexed: 02/26/2023]
Abstract
BACKGROUND Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.
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Affiliation(s)
- Mathijs Willemsen
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - John S Barber
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - Erika Van Nieuwenhove
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium; Department of Pediatrics, University Hospitals Leuven, Leuven, Belgium
| | - Frederik Staels
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; Department of Microbiology, Immunology, and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium
| | - Margaux Gerbaux
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; Pediatric Department, Academic Children Hospital Queen Fabiola, Université Libre de Bruxelles, Brussels, Belgium
| | - Julika Neumann
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - Teresa Prezzemolo
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - Emanuela Pasciuto
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - Vasiliki Lagou
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium
| | - Nancy Boeckx
- Department of Laboratory Medicine, University Hospitals Leuven, Leuven, Belgium
| | - Jessica Filtjens
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuve, Belgium
| | - Amber De Visscher
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuve, Belgium
| | - Patrick Matthys
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Immunobiology, Rega Institute for Medical Research, KU Leuven, Leuve, Belgium
| | - Rik Schrijvers
- Department of Microbiology, Immunology, and Transplantation, Allergy and Clinical Immunology Research Group, KU Leuven, Leuven, Belgium
| | - Thomas Tousseyn
- Department of Pathology, University Hospitals Leuven, Leuven, Belgium
| | - Mark O'Driscoll
- Human DNA Damage Response Disorders Group, Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom
| | - Giorgia Bucciol
- Department of Microbiology, Immunology, and Transplantation, Laboratory for Inborn Errors of Immunity, KU Leuven, Leuven, Belgium; Department of Pediatrics, Division of Primary Immunodeficiencies, University Hospitals Leuven, Leuven
| | - Susan Schlenner
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium
| | - Isabelle Meyts
- Department of Microbiology, Immunology, and Transplantation, Laboratory for Inborn Errors of Immunity, KU Leuven, Leuven, Belgium; Department of Pediatrics, Division of Primary Immunodeficiencies, University Hospitals Leuven, Leuven.
| | - Stephanie Humblet-Baron
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium.
| | - Adrian Liston
- Department of Microbiology, Immunology, and Transplantation, Laboratory of Adaptive Immunity, KU Leuven, Leuven, Belgium; VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium; Immunology Programme, The Babraham Institute, Babraham Research Campus, Cambridge, United Kingdom.
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3
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Abd Wahab S, Remus D. Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53. eLife 2020; 9:58571. [PMID: 32701054 PMCID: PMC7398698 DOI: 10.7554/elife.58571] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2020] [Accepted: 07/22/2020] [Indexed: 12/12/2022] Open
Abstract
Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.
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Affiliation(s)
- Syafiq Abd Wahab
- Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States.,Weill-Cornell Graduate School of Medical Sciences, New York, United States
| | - Dirk Remus
- Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States.,Weill-Cornell Graduate School of Medical Sciences, New York, United States
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4
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Wahab SA, Remus D. Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53.. [DOI: 10.1101/2020.05.04.077628] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
Abstract
ABSTRACTEukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.
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Knockleby J, Kim BJ, Mehta A, Lee H. Cdk1-mediated phosphorylation of Cdc7 suppresses DNA re-replication. Cell Cycle 2016; 15:1494-505. [PMID: 27105124 PMCID: PMC4934051 DOI: 10.1080/15384101.2016.1176658] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2016] [Revised: 03/23/2016] [Accepted: 04/06/2016] [Indexed: 12/18/2022] Open
Abstract
To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1α associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1α/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.
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Affiliation(s)
- James Knockleby
- Tumour Biology Group, Advanced Medical Research Institute of Canada, Health Sciences North, Sudbury, Ontario, Canada
| | - Byung Ju Kim
- Tumour Biology Group, Advanced Medical Research Institute of Canada, Health Sciences North, Sudbury, Ontario, Canada
| | - Avani Mehta
- Tumour Biology Group, Advanced Medical Research Institute of Canada, Health Sciences North, Sudbury, Ontario, Canada
| | - Hoyun Lee
- Tumour Biology Group, Advanced Medical Research Institute of Canada, Health Sciences North, Sudbury, Ontario, Canada
- Departments of Medicine, the Faculty of Medicine, the University of Ottawa, Ottawa, Ontario, Canada
- Northern Ontario School of Medicine, Sudbury, Ontario, Canada
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6
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Zhang M, Sun F, Chen F, Zhou B, Duan Y, Su H, Lin X. Subcellular proteomic approach for identifying the signaling effectors of protein kinase C-β₂ under high glucose conditions in human umbilical vein endothelial cells. Mol Med Rep 2015; 12:7247-62. [PMID: 26459836 PMCID: PMC4626174 DOI: 10.3892/mmr.2015.4403] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2014] [Accepted: 08/05/2015] [Indexed: 11/06/2022] Open
Abstract
The high glucose‑induced activation of protein kinase C‑β2 (PKC‑β2) has an essential role in the pathophysiology of diabetes‑associated vascular disease. In the present study, human umbilical vein endothelial cells (HUVECs) were cultured in high and normal glucose conditions prior to being infected with a recombinant adenovirus to induce the overexpression of PKC‑β2. The activity of PKC‑β2 was also decreased using a selective PKC‑β2 inhibitor. A series of two‑dimensional electrophoresis images detected ~800 spots in the nuclei, and ~600 spots in the cytosol. Following intra‑ and inter‑group cross‑matching, 38 significantly altered spots were identified as high glucose‑induced and PKC‑β2‑associated nuclear proteins. In addition to the observation that the regulation of key proteins involved in the nuclear factor (NF)‑κB signaling cascade occurred in the cytosol, various transcription factors, including peroxisome proliferator‑activated receptor δ (PPAR‑δ), were also altered in the nuclei. A human protein‑protein interaction network of potential connections of PKC‑β2‑associated proteins was constructed in the proteomics investigation using Biological General Repository for Interaction Datasets. The results indicated that PKC‑β2 may be involved in high glucose‑induced glucose and lipid crosstalk by regulating PPAR‑δ. In addition, NF‑κB inhibitor‑interacting Ras‑like protein 1 may be important in the PKC‑β2‑NF‑κB inhibitor‑NF‑κB signaling pathway in HUVECs under high‑glucose conditions.
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Affiliation(s)
- Min Zhang
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
| | - Fang Sun
- Department of Hypertension and Endocrinology, Daping Hospital, Third Military Medical University, Chongqing 400042, P.R. China
| | - Fangfang Chen
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
| | - Bo Zhou
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
| | - Yaqian Duan
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
| | - Hong Su
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
| | - Xuebo Lin
- Department of Endocrinology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P.R. China
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7
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Stephenson R, Hosler MR, Gavande NS, Ghosh AK, Weake VM. Characterization of a Drosophila ortholog of the Cdc7 kinase: a role for Cdc7 in endoreplication independent of Chiffon. J Biol Chem 2014; 290:1332-47. [PMID: 25451925 DOI: 10.1074/jbc.m114.597948] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cdc7 is a serine-threonine kinase that phosphorylates components of the pre-replication complex during DNA replication initiation. Cdc7 is highly conserved, and Cdc7 orthologs have been characterized in organisms ranging from yeast to humans. Cdc7 is activated specifically during late G1/S phase by binding to its regulatory subunit, Dbf4. Drosophila melanogaster contains a Dbf4 ortholog, Chiffon, which is essential for chorion amplification in Drosophila egg chambers. However, no Drosophila ortholog of Cdc7 has yet been characterized. Here, we report the functional and biochemical characterization of a Drosophila ortholog of Cdc7. Co-expression of Drosophila Cdc7 and Chiffon is able to complement a growth defect in yeast containing a temperature-sensitive Cdc7 mutant. Cdc7 and Chiffon physically interact and can be co-purified from insect cells. Cdc7 phosphorylates the known Cdc7 substrates Mcm2 and histone H3 in vitro, and Cdc7 kinase activity is stimulated by Chiffon and inhibited by the Cdc7-specific inhibitor XL413. Drosophila egg chamber follicle cells deficient for Cdc7 have a defect in two types of DNA replication, endoreplication and chorion gene amplification. However, follicle cells deficient for Chiffon have a defect in chorion gene amplification but still undergo endocycling. Our results show that Cdc7 interacts with Chiffon to form a functional Dbf4-dependent kinase complex and that Cdc7 is necessary for DNA replication in Drosophila egg chamber follicle cells. Additionally, we show that Chiffon is a member of an expanding subset of DNA replication initiation factors that are not strictly required for endoreplication in Drosophila.
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Affiliation(s)
| | | | | | - Arun K Ghosh
- Chemistry and Medicinal Chemistry, and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907
| | - Vikki M Weake
- From the Departments of Biochemistry and Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907
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8
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Poh WT, Chadha GS, Gillespie PJ, Kaldis P, Blow JJ. Xenopus Cdc7 executes its essential function early in S phase and is counteracted by checkpoint-regulated protein phosphatase 1. Open Biol 2014; 4:130138. [PMID: 24403013 PMCID: PMC3909274 DOI: 10.1098/rsob.130138] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2013] [Accepted: 12/03/2013] [Indexed: 01/31/2023] Open
Abstract
The initiation of DNA replication requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase Cdk activity has been intensively studied, relatively little is known about how Cdc7 regulates progression through S phase. We have used a Cdc7 inhibitor, PHA-767491, to dissect the role of Cdc7 in Xenopus egg extracts. We show that hyperphosphorylation of mini-chromosome maintenance (MCM) proteins by Cdc7 is required for the initiation, but not for the elongation, of replication forks. Unlike Cdks, we demonstrate that Cdc7 executes its essential functions by phosphorylating MCM proteins at virtually all replication origins early in S phase and is not limiting for progression through the Xenopus replication timing programme. We demonstrate that protein phosphatase 1 (PP1) is recruited to chromatin and rapidly reverses Cdc7-mediated MCM hyperphosphorylation. Checkpoint kinases induced by DNA damage or replication inhibition promote the association of PP1 with chromatin and increase the rate of MCM dephosphorylation, thereby counteracting the previously completed Cdc7 functions and inhibiting replication initiation. This novel mechanism for regulating Cdc7 function provides an explanation for previous contradictory results concerning the control of Cdc7 by checkpoint kinases and has implications for the use of Cdc7 inhibitors as anti-cancer agents.
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Affiliation(s)
- Wei Theng Poh
- Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
- Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), Singapore 138673, Republic of Singapore
| | - Gaganmeet Singh Chadha
- Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
| | - Peter J. Gillespie
- Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
| | - Philipp Kaldis
- Institute of Molecular and Cell Biology, Agency for Science, Technology, and Research (A*STAR), Singapore 138673, Republic of Singapore
| | - J. Julian Blow
- Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow St., Dundee DD1 5EH, UK
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Abstract
DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.
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Affiliation(s)
- Khalid Siddiqui
- Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts EN6 3LD, United Kingdom
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10
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Gillespie PJ, Gambus A, Blow JJ. Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. Methods 2012; 57:203-13. [PMID: 22521908 PMCID: PMC3437562 DOI: 10.1016/j.ymeth.2012.03.029] [Citation(s) in RCA: 57] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2011] [Revised: 03/23/2012] [Accepted: 03/24/2012] [Indexed: 12/20/2022] Open
Abstract
The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.
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Affiliation(s)
- Peter J. Gillespie
- Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK
| | - Agnieszka Gambus
- School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
| | - J. Julian Blow
- Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee, Dow Street, Dundee DD1 5EH, UK
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11
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Sasaki T, Li A, Gillespie PJ, Blow JJ, Gilbert DM. Evidence for a mammalian late-G1 phase inhibitor of replication licensing distinct from geminin or Cdk activity. Nucleus 2011; 2:455-64. [PMID: 21983086 DOI: 10.4161/nucl.2.5.17859] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Pre-replication complexes (pre-RCs) are assembled onto DNA during late mitosis and G1 to license replication origins for use in S phase. In order to prevent re-replication of DNA, licensing must be completely shutdown prior to entry into S phase. While mechanisms preventing re-replication during S phase and mitosis have been elucidated, the means by which cells first prevent licensing during late G1 phase are poorly understood. We have employed a hybrid mammalian / Xenopus egg extract replication system to dissect activities that inhibit replication licensing at different stages of the cell cycle in Chinese Hamster Ovary (CHO) cells. We find that soluble extracts from mitotic cells inhibit licensing through a combination of geminin and Cdk activities, while extracts from S-phase cells inhibit licensing predominantly through geminin alone. Surprisingly however, geminin did not accumulate until after cells enter S phase. Unlike extracts from cells in early G1 phase, extracts from late G1 phase and early S phase cells contained an inhibitor of licensing that could not be accounted for by either geminin or Cdk. Moreover, inhibiting cyclin and geminin protein synthesis or inhibiting Cdk activity early in G1 phase did not prevent the appearance of inhibitory activity. These results suggest that a soluble inhibitor of replication licensing appears prior to entry into S phase that is distinct from either geminin or Cdk activity. Our hybrid system should permit the identification of this and other novel cell cycle regulatory activities.
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Affiliation(s)
- Takayo Sasaki
- Department of Biological Science, Florida State University, Tallahassee, FL, USA
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12
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Mackenzie NCW, Lillico SG, Brown K, Wolf CR, Whitelaw CBA. Evaluation of RNA-knockdown strategies for modulation of cytochrome P450 reductase activity in mouse hepatocytes. JOURNAL OF RNAI AND GENE SILENCING : AN INTERNATIONAL JOURNAL OF RNA AND GENE TARGETING RESEARCH 2010; 6:416-21. [PMID: 21350684 PMCID: PMC3043560] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/05/2010] [Revised: 11/02/2010] [Accepted: 11/18/2010] [Indexed: 11/02/2022]
Abstract
Transgenic technologies can provide important animal models for studying drug-metabolizing enzymes. Our overall aim was to generate versatile cell and animal systems that exhibited varying levels of cytochrome P450 oxidoreductase (POR) activity, more accurately modelling the human population for pharmacological and toxicology studies. Towards this goal we evaluated RNA-interference constructs designed for use in vitro and in vivo for reducing POR activity in hepatocytes. This study clearly demonstrates that both POR protein level and reductase activity can be significantly knocked down in Hepa-1 cells in vitro, while highlighting the difficulty in predicting knockdown efficiency in transgenic animals. The high levels of embryonic lethality observed, and inability to produce multi-copy transgenic animals indicates that high levels of shRNA expression may be detrimental to embryonic development.
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Affiliation(s)
- Neil CW Mackenzie
- Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, UK
| | - Simon G Lillico
- Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, UK
| | - Ken Brown
- CXR Biosciences Limited, James Lindsay Place, Dundee Technopole, Dundee, DD1 5JJ, UK
| | - Charles R Wolf
- CXR Biosciences Limited, James Lindsay Place, Dundee Technopole, Dundee, DD1 5JJ, UK,University of Dundee Biomedical Research Institute, Ninewells Hospital and Medical School, Dundee, DD1 9SY, UK
| | - Christopher BA Whitelaw
- Division of Developmental Biology, The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian, EH25 9PS, UK,Correspondence to: Neil Mackenzie, , Tel: +44 (0)131 5274200, Fax: +44 (0)131 4400434
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13
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Kundu LR, Kumata Y, Kakusho N, Watanabe S, Furukohri A, Waga S, Seki M, Masai H, Enomoto T, Tada S. Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex. Nucleic Acids Res 2010; 38:5409-18. [PMID: 20421204 PMCID: PMC2938227 DOI: 10.1093/nar/gkq262] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022] Open
Abstract
Mcm2–7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2–7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2–4–6–7 in purified systems, rather modulates Mcm2–7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2–7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.
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Affiliation(s)
- Lena R Kundu
- Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan
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14
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Hughes S, Jenkins V, Dar MJ, Engelman A, Cherepanov P. Transcriptional co-activator LEDGF interacts with Cdc7-activator of S-phase kinase (ASK) and stimulates its enzymatic activity. J Biol Chem 2009; 285:541-54. [PMID: 19864417 DOI: 10.1074/jbc.m109.036491] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Lens epithelium-derived growth factor (LEDGF) is an important co-factor of human immunodeficiency virus DNA integration; however, its cellular functions are poorly characterized. We now report identification of the Cdc7-activator of S-phase kinase (ASK) heterodimer as a novel interactor of LEDGF. Both kinase subunits co-immunoprecipitated with endogenous LEDGF from human cell extracts. Truncation analyses identified the integrase-binding domain of LEDGF as essential and minimally sufficient for the interaction with Cdc7-ASK. Reciprocally, the interaction required autophosphorylation of the kinase and the presence of 50 C-terminal residues of ASK. The kinase phosphorylated LEDGF in vitro, with Ser-206 being the major target, and LEDGF phosphorylated at this residue could be detected during S phase of the cell cycle. LEDGF potently stimulated the enzymatic activity of Cdc7-ASK, increasing phosphorylation of MCM2 in vitro by more than 10-fold. This enzymatic stimulation as well as phosphorylation of LEDGF depended on the protein-protein interaction. Intriguingly, removing the C-terminal region of ASK, involved in the interaction with LEDGF, resulted in a hyperactive kinase. Our results indicate that the interaction with LEDGF relieves autoinhibition of Cdc7-ASK kinase, imposed by the C terminus of ASK.
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Affiliation(s)
- Siobhan Hughes
- Division of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, United Kingdom and
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15
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Bruck I, Kaplan D. Dbf4-Cdc7 phosphorylation of Mcm2 is required for cell growth. J Biol Chem 2009; 284:28823-31. [PMID: 19692334 DOI: 10.1074/jbc.m109.039123] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Delta); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.
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Affiliation(s)
- Irina Bruck
- Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37235, USA
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16
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Francis LI, Randell JCW, Takara TJ, Uchima L, Bell SP. Incorporation into the prereplicative complex activates the Mcm2-7 helicase for Cdc7-Dbf4 phosphorylation. Genes Dev 2009; 23:643-54. [PMID: 19270162 DOI: 10.1101/gad.1759609] [Citation(s) in RCA: 106] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The essential S-phase kinase Cdc7-Dbf4 acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2-7 replicative helicase. Also known as Dbf4-dependent kinase (DDK), this kinase preferentially targets chromatin-associated Mcm2-7 complexes that are assembled on the DNA during prereplicative complex (pre-RC) formation. Here we address the mechanisms that control the specificity of DDK action. We show that incorporation of Mcm2-7 into the pre-RC increased the level and changes the specificity of DDK phosphorylation of this complex. In the context of the pre-RC, DDK preferentially targets a conformationally distinct subpopulation of Mcm2-7 complexes that is tightly linked to the origin DNA. This targeting requires DDK to tightly associate with Mcm2-7 complexes in a Dbf4-dependent manner. Importantly, we find that DDK association with and phosphorylation of origin-linked Mcm2-7 complexes require prior phosphorylation of the pre-RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the origin-bound Mcm2-7 complexes that will drive replication fork movement during S phase and suggest new mechanisms to regulate origin activity.
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Affiliation(s)
- Laura I Francis
- Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
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17
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Takahashi TS, Basu A, Bermudez V, Hurwitz J, Walter JC. Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts. Genes Dev 2008; 22:1894-905. [PMID: 18628396 PMCID: PMC2492736 DOI: 10.1101/gad.1683308] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2008] [Accepted: 05/23/2008] [Indexed: 12/23/2022]
Abstract
To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.
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Affiliation(s)
- Tatsuro S. Takahashi
- Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Abhijit Basu
- Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
| | - Vladimir Bermudez
- Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
| | - Jerard Hurwitz
- Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA
| | - Johannes C. Walter
- Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
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18
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Silva T, Bradley RH, Gao Y, Coue M. Xenopus CDC7/DRF1 complex is required for the initiation of DNA replication. J Biol Chem 2006; 281:11569-76. [PMID: 16507577 DOI: 10.1074/jbc.m510278200] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Cdc7 kinase is essential for the initiation of DNA replication in eukaryotes. Two regulatory subunits of the Xenopus Cdc7 kinase have been identified: XDbf4 and XDrf1. In this study we determined the expression pattern of XDbf4 and XDrf1 and examined their involvement in DNA replication. We show that XDrf1 expression is restricted to oogenesis and early embryos, whereas XDbf4 is expressed throughout development. Immunodepletion from Xenopus egg extracts indicated that both proteins are only found in complexes with XCdc7 and there is a 5-fold molar excess of the XCdc7/Drf1 over SCdc7/Dbf4 complexes. Both complexes exhibit kinase activity and are differentially phosphorylated during the cell cycle. Depletion of the XCdc7/Drf1 from egg extracts inhibited DNA replication, whereas depletion of XCdc7/Dbf4 had little effect. Chromatin binding studies indicated that XCdc7/Drf1 is required for pre-replication complex activation but not their assembly. XCdc7/Dbf4 complexes bound to the chromatin in two steps: the first step was independent of pre-replication complex assembly and the second step was dependent on pre-replication complex activation. By contrast, binding of XCdc7/Drf1 complexes was entirely dependent on pre-replication complex assembly. Finally, we present evidence that the association of the two complexes on the chromatin is not regulated by ATR checkpoint pathways that result from DNA replication blocks. These data suggest that Cdc7/Drf1 but not Cdc7/Dbf4 complexes support the initiation of DNA replication in Xenopus egg extracts and during early embryonic development.
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Affiliation(s)
- Tania Silva
- Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA
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19
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Takahashi TS, Walter JC. Cdc7-Drf1 is a developmentally regulated protein kinase required for the initiation of vertebrate DNA replication. Genes Dev 2005; 19:2295-300. [PMID: 16204181 PMCID: PMC1240038 DOI: 10.1101/gad.1339805] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Cdc7, a protein kinase required for the initiation of eukaryotic DNA replication, is activated by a regulatory subunit, Dbf4. A second activator of Cdc7 called Drf1 exists in vertebrates, but its function is unknown. Here, we report that in Xenopus egg extracts, Cdc7-Drf1 is far more abundant than Cdc7-Dbf4, and removal of Drf1 but not Dbf4 severely inhibits phosphorylation of Mcm4 and DNA replication. After gastrulation, when the cell cycle acquires somatic characteristics, Drf1 levels decline sharply and Cdc7-Dbf4 becomes the more abundant kinase. These results identify Drf1 as a developmentally regulated, essential activator of Cdc7 in Xenopus.
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Affiliation(s)
- Tatsuro S Takahashi
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA
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20
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Guo B, Romero J, Kim BJ, Lee H. High levels of Cdc7 and Dbf4 proteins can arrest cell-cycle progression. Eur J Cell Biol 2005; 84:927-38. [PMID: 16325502 DOI: 10.1016/j.ejcb.2005.09.016] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2005] [Revised: 09/07/2005] [Accepted: 09/08/2005] [Indexed: 10/25/2022] Open
Abstract
Cdc7-Dbf4 serine/threonine kinase is essential for initiation of DNA replication. It was previously found that overexpression of certain replication proteins such as Cdc6 and Cdt1 in fission yeast resulted in multiple rounds of DNA replication in the absence of mitosis. Since this phenomenon is dependent upon the presence of wild-type Cdc7/Hsk1, we hypothesized that high levels of Cdc7 and/or Dbf4 could also cause multiple rounds of DNA replication, or could facilitate entry into S phase. To test this hypothesis, we transiently overexpressed hamster Cdc7, Dbf4 or both in CHO cells. Direct observations of individual cells by fluorescence microscopy and flow cytometric analysis on cell populations suggest that overexpression of Cdc7 and/or Dbf4 does not result in multiple rounds of DNA replication or facilitating entry into S phase. In contrast, moderately increased levels of Dbf4, but not Cdc7, cause cell-cycle arrest in G2/M. This G2/M arrest coincides with hyperphosphorylation of Cdc2/Cdk1 at Tyr-15, raising the possibility that high levels of Dbf4 may activate a G2/M cell-cycle checkpoint. Further increase in Cdc7 and/or Dbf4 by 2-4 fold can arrest cells in G1 and significantly slow down S-phase progression for the cells already in S phase.
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Affiliation(s)
- Baoqing Guo
- Department of Research, Northeastern Ontario Regional Cancer Centre, Sudbury, Canada
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21
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Varrin AE, Prasad AA, Scholz RP, Ramer MD, Duncker BP. A mutation in Dbf4 motif M impairs interactions with DNA replication factors and confers increased resistance to genotoxic agents. Mol Cell Biol 2005; 25:7494-504. [PMID: 16107698 PMCID: PMC1190303 DOI: 10.1128/mcb.25.17.7494-7504.2005] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Dbf4/Cdc7 is required for DNA replication in Saccharomyces cerevisiae and appears to be a target in the S-phase checkpoint. Previously, a 186-amino-acid Dbf4 region that mediates interactions with both the origin recognition complex and Rad53 was identified. We now show this domain also mediates the association between Dbf4 and Mcm2, a key Dbf4/Cdc7 phosphorylation target. Two conserved sequences, the N and M motifs, have been identified within this Dbf4 region. Removing motif M (Dbf4DeltaM) impairs the ability of Dbf4 to support normal cell cycle progression and abrogates the Dbf4-Mcm2 association but has no effect on the Dbf4-Rad53 interaction. In contrast, deleting motif N (Dbf4DeltaN) does not affect the essential function of Dbf4, disrupts the Dbf4-Rad53 interaction, largely preserves the Dbf4-Mcm2 association, and renders the cells hypersensitive to genotoxic agents. Surprisingly, Dbf4DeltaM interacts strongly with Orc2, while Dbf4DeltaN does not. The DBF4 allele dna52-1 was cloned and sequenced, revealing a single point mutation within the M motif. This mutant is unable to maintain interactions with either Mcm2 or Orc2 at the semipermissive temperature of 30 degrees C, while the interaction with Rad53 is preserved. Furthermore, this mutation confers increased resistance to genotoxic agents, which we propose is more likely due to a role for Dbf4 in the resumption of fork progression following checkpoint-induced arrest than prevention of late origin firing. Thus, the alteration of the M motif may facilitate the role of Dbf4 as a checkpoint target.
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Affiliation(s)
- Angela E Varrin
- Department of Biology, University of Waterloo, Ontario, Canada
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22
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Abstract
Initiation and completion of DNA replication defines the beginning and ending of S phase of the cell cycle. Successful progression through S phase requires that replication be properly regulated and monitored to ensure that the entire genome is duplicated exactly once, without errors, in a timely fashion. Given the immense size and complexity of eukaryotic genomes, this presents a significant challenge for the cell. As a result, DNA replication has evolved into a tightly regulated process involving the coordinated action of numerous factors that function in all phases of the cell cycle. We will review our current understanding of these processes from the formation of prereplicative complexes in preparation for S phase to the series of events that culminate in the loading of DNA polymerases during S phase. We will incorporate structural data from archaeal and bacterial replication proteins and discuss their implications for understanding the mechanism of action of their corresponding eukaryotic homologues. We will also describe the concept of replication licensing which protects against genomic instability by limiting initiation events to once per cell cycle. Lastly, we will review our knowledge of checkpoint pathways that maintain the integrity of stalled forks and relay defects in replication to the rest of the cell cycle.
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Affiliation(s)
- David Y Takeda
- Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
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23
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Ferenbach A, Li A, Brito-Martins M, Blow JJ. Functional domains of the Xenopus replication licensing factor Cdt1. Nucleic Acids Res 2005; 33:316-24. [PMID: 15653632 PMCID: PMC546161 DOI: 10.1093/nar/gki176] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2004] [Revised: 12/20/2004] [Accepted: 12/20/2004] [Indexed: 12/21/2022] Open
Abstract
During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.
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Affiliation(s)
- Andrew Ferenbach
- Wellcome Trust Biocentre, University of Dundee Dow Street, Dundee DD1 5EH, UK
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24
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Li A, Blow JJ. Cdt1 downregulation by proteolysis and geminin inhibition prevents DNA re-replication in Xenopus. EMBO J 2004; 24:395-404. [PMID: 15616577 PMCID: PMC545810 DOI: 10.1038/sj.emboj.7600520] [Citation(s) in RCA: 108] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2004] [Accepted: 11/23/2004] [Indexed: 01/04/2023] Open
Abstract
In late mitosis and G1, Mcm2-7 are assembled onto replication origins to 'license' them for initiation. At other cell cycle stages, licensing is inhibited, thus ensuring that origins fire only once per cell cycle. Three additional factors--the origin recognition complex, Cdc6 and Cdt1--are required for origin licensing. We examine here how licensing is regulated in Xenopus egg extracts. We show that Cdt1 is downregulated late in the cell cycle by two different mechanisms: proteolysis, which occurs in part due to the activity of the anaphase-promoting complex (APC/C), and inhibition by a protein called geminin. If both these regulatory mechanisms are abrogated, extracts undergo uncontrolled re-licensing and re-replication. The extent of re-replication is limited by checkpoint kinases that are activated as a consequence of re-replication itself. These results allow us to build a comprehensive model of how re-replication of DNA is prevented in Xenopus, with Cdt1 regulation being the key feature. The results also explain the original experiments that led to the proposal of a replication licensing factor.
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Affiliation(s)
- Anatoliy Li
- Wellcome Trust Biocentre, University of Dundee, Dundee, UK
| | - J Julian Blow
- Wellcome Trust Biocentre, University of Dundee, Dundee, UK
- Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: +44 1382 345797; Fax: +44 1382 348072; E-mail:
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