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Ma C, Gurkan-Cavusoglu E. A comprehensive review of computational cell cycle models in guiding cancer treatment strategies. NPJ Syst Biol Appl 2024; 10:71. [PMID: 38969664 PMCID: PMC11226463 DOI: 10.1038/s41540-024-00397-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 06/24/2024] [Indexed: 07/07/2024] Open
Abstract
This article reviews the current knowledge and recent advancements in computational modeling of the cell cycle. It offers a comparative analysis of various modeling paradigms, highlighting their unique strengths, limitations, and applications. Specifically, the article compares deterministic and stochastic models, single-cell versus population models, and mechanistic versus abstract models. This detailed analysis helps determine the most suitable modeling framework for various research needs. Additionally, the discussion extends to the utilization of these computational models to illuminate cell cycle dynamics, with a particular focus on cell cycle viability, crosstalk with signaling pathways, tumor microenvironment, DNA replication, and repair mechanisms, underscoring their critical roles in tumor progression and the optimization of cancer therapies. By applying these models to crucial aspects of cancer therapy planning for better outcomes, including drug efficacy quantification, drug discovery, drug resistance analysis, and dose optimization, the review highlights the significant potential of computational insights in enhancing the precision and effectiveness of cancer treatments. This emphasis on the intricate relationship between computational modeling and therapeutic strategy development underscores the pivotal role of advanced modeling techniques in navigating the complexities of cell cycle dynamics and their implications for cancer therapy.
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Affiliation(s)
- Chenhui Ma
- Department of Electrical, Computer and Systems Engineering, Case Western Reserve University, Cleveland, OH, USA.
| | - Evren Gurkan-Cavusoglu
- Department of Electrical, Computer and Systems Engineering, Case Western Reserve University, Cleveland, OH, USA
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2
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Li C, Tan Y, Ma X, Wang Z, Meng T, Sun Q. CDT1 is the major functional regulatory subunit of the pre-replication complex in zygotes. Cell Prolif 2022; 56:e13377. [PMID: 36479743 PMCID: PMC9977660 DOI: 10.1111/cpr.13377] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 11/08/2022] [Accepted: 11/23/2022] [Indexed: 12/13/2022] Open
Abstract
Pre-replication complex (pre-RC) is critical for DNA replication initiation. CDT1 and MCM2 are the subunits of pre-RC, and proper regulation of CDT1 and MCM2 are necessary for DNA replication and cell proliferation. The present study aimed to explore the role of CDT1 and MCM2 in oocyte meiotic maturation and early embryonic development. The depletion and overexpression of Cdt1 and Mcm2 in oocyte and zygote were achieved by microinjecting specific siRNA and mRNA to explored their functions in oocyte meiotic maturation and embryonic development. Then, we examined the effect of CDT1 and MCM2 on other signal pathways by immunostaining the expression of related maker genes. We showed that neither depletion nor overexpression of Cdt1 affected oocyte meiotic progressions. The CDT1 was degraded in S phase and remained at a low level in G2 phase of zygote. Exogenous expression of Cdt1 in G2 phase led to embryo attest at zygote stage. Mechanistically, CDT1 overexpression induced DNA re-replication and thus DNA damage check-point activation. Protein abundance of MCM2 was stable throughout the cell cycle, and embryos with overexpressed MCM2 could develop to blastocysts normally. Overexpression or depletion of Mcm2 also had no effect on oocyte meiotic maturation. Our results indicate that pre-RC subunits CDT1 and MCM2 are not involved in oocyte meiotic maturation. In zygote, CDT1 but not MCM2 is the major regulator of DNA replication in a cell cycle dependent manner. Furthermore, its' degradation is essential for zygotes to prevent from DNA re-replication in G2 stage.
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Affiliation(s)
- Chao Li
- State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of Zoology, Chinese Academy of SciencesBeijingChina,University of Chinese Academy of SciencesBeijingChina
| | - Yong‐Peng Tan
- Fertility Preservation Lab, Guangdong‐Hong Kong Metabolism & Reproduction Joint LaboratoryReproductive Medicine Center, Guangdong Second Provincial General HospitalGuangzhouChina
| | - Xue‐Shan Ma
- Reproductive Genetics DepartmentThe Affiliated Tai'an City Central Hospital of Qingdao UniversityTaianChina
| | - Zhen‐Bo Wang
- State Key Laboratory of Stem Cell and Reproductive BiologyInstitute of Zoology, Chinese Academy of SciencesBeijingChina,University of Chinese Academy of SciencesBeijingChina
| | - Tie‐Gang Meng
- Fertility Preservation Lab, Guangdong‐Hong Kong Metabolism & Reproduction Joint LaboratoryReproductive Medicine Center, Guangdong Second Provincial General HospitalGuangzhouChina
| | - Qing‐Yuan Sun
- Fertility Preservation Lab, Guangdong‐Hong Kong Metabolism & Reproduction Joint LaboratoryReproductive Medicine Center, Guangdong Second Provincial General HospitalGuangzhouChina
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3
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Reusswig KU, Bittmann J, Peritore M, Courtes M, Pardo B, Wierer M, Mann M, Pfander B. Unscheduled DNA replication in G1 causes genome instability and damage signatures indicative of replication collisions. Nat Commun 2022; 13:7014. [PMID: 36400763 PMCID: PMC9674678 DOI: 10.1038/s41467-022-34379-2] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2021] [Accepted: 10/24/2022] [Indexed: 11/19/2022] Open
Abstract
DNA replicates once per cell cycle. Interfering with the regulation of DNA replication initiation generates genome instability through over-replication and has been linked to early stages of cancer development. Here, we engineer genetic systems in budding yeast to induce unscheduled replication in a G1-like cell cycle state. Unscheduled G1 replication initiates at canonical S-phase origins. We quantifiy the composition of replisomes in G1- and S-phase and identified firing factors, polymerase α, and histone supply as factors that limit replication outside S-phase. G1 replication per se does not trigger cellular checkpoints. Subsequent replication during S-phase, however, results in over-replication and leads to chromosome breaks and chromosome-wide, strand-biased occurrence of RPA-bound single-stranded DNA, indicating head-to-tail replication collisions as a key mechanism generating genome instability upon G1 replication. Low-level, sporadic induction of G1 replication induces an identical response, indicating findings from synthetic systems are applicable to naturally occurring scenarios of unscheduled replication initiation.
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Affiliation(s)
- Karl-Uwe Reusswig
- grid.418615.f0000 0004 0491 845XDNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany ,grid.38142.3c000000041936754XPresent Address: Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115 USA ,grid.65499.370000 0001 2106 9910Present Address: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215 USA
| | - Julia Bittmann
- grid.418615.f0000 0004 0491 845XDNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Martina Peritore
- grid.418615.f0000 0004 0491 845XDNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany ,grid.7551.60000 0000 8983 7915Present Address: Genome Maintenance Mechanisms in Health and Disease, Institute of Aerospace Medicine, German Aerospace Center (DLR), 51147 Cologne, Germany
| | - Mathilde Courtes
- grid.433120.7Institut de Génétique Humaine (IGH), Université de Montpellier – Centre National de la Recherche Scientifique, 34396 Montpellier, France
| | - Benjamin Pardo
- grid.433120.7Institut de Génétique Humaine (IGH), Université de Montpellier – Centre National de la Recherche Scientifique, 34396 Montpellier, France
| | - Michael Wierer
- grid.418615.f0000 0004 0491 845XProteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany ,grid.5254.60000 0001 0674 042XPresent Address: Proteomics Research Infrastructure, University of Copenhagen, 2200 Copenhagen, Denmark
| | - Matthias Mann
- grid.418615.f0000 0004 0491 845XProteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany
| | - Boris Pfander
- grid.418615.f0000 0004 0491 845XDNA Replication and Genome Integrity, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany ,grid.7551.60000 0000 8983 7915Present Address: Genome Maintenance Mechanisms in Health and Disease, Institute of Aerospace Medicine, German Aerospace Center (DLR), 51147 Cologne, Germany ,grid.6190.e0000 0000 8580 3777Present Address: Genome Maintenance Mechanisms in Health and Disease, Institute of Genome Stability in Ageing and Disease, CECAD Research Center, University of Cologne, 50931 Cologne, Germany
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4
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Limas JC, Littlejohn AN, House AM, Kedziora KM, Mouery BL, Ma B, Fleifel D, Walens A, Aleman MM, Dominguez D, Cook JG. Quantitative profiling of adaptation to cyclin E overproduction. Life Sci Alliance 2022; 5:e202201378. [PMID: 35173014 PMCID: PMC8860095 DOI: 10.26508/lsa.202201378] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2022] [Revised: 01/28/2022] [Accepted: 01/31/2022] [Indexed: 01/03/2023] Open
Abstract
Cyclin E/CDK2 drives cell cycle progression from G1 to S phase. Despite the toxicity of cyclin E overproduction in mammalian cells, the cyclin E gene is overexpressed in some cancers. To further understand how cells can tolerate high cyclin E, we characterized non-transformed epithelial cells subjected to chronic cyclin E overproduction. Cells overproducing cyclin E, but not cyclins D or A, briefly experienced truncated G1 phases followed by a transient period of DNA replication origin underlicensing, replication stress, and impaired proliferation. Individual cells displayed substantial intercellular heterogeneity in cell cycle dynamics and CDK activity. Each phenotype improved rapidly despite high cyclin E-associated activity. Transcriptome analysis revealed adapted cells down-regulated a cohort of G1-regulated genes. Withdrawing cyclin E from adapted cells only partially reversed underlicensing indicating that adaptation is at least partly non-genetic. This study provides evidence that mammalian cyclin E/CDK inhibits origin licensing indirectly through premature S phase onset and provides mechanistic insight into the relationship between CDKs and licensing. It serves as an example of oncogene adaptation that may recapitulate molecular changes during tumorigenesis.
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Affiliation(s)
- Juanita C Limas
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Amiee N Littlejohn
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Amy M House
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Katarzyna M Kedziora
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Bioinformatics and Analytics Research Collaborative (BARC), University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Brandon L Mouery
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Boyang Ma
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Dalia Fleifel
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Andrea Walens
- Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Maria M Aleman
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Daniel Dominguez
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
| | - Jeanette Gowen Cook
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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5
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Johnson MC, Can G, Santos MM, Alexander D, Zegerman P. Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase. eLife 2021; 10:e63589. [PMID: 33399537 PMCID: PMC7806266 DOI: 10.7554/elife.63589] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 01/04/2021] [Indexed: 01/02/2023] Open
Abstract
Checkpoints maintain the order of cell cycle events during DNA damage or incomplete replication. How the checkpoint response is tailored to different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in budding yeast occurs by Rad53-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed, we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M, preventing gene amplification. In addition, we show that inhibition of Sld3 and Dbf4 in G1 prevents premature initiation at all origins at the G1/S transition. This study redefines the scope of the 'S-phase checkpoint' with implications for understanding checkpoint function in cancers that lack cell cycle controls.
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Affiliation(s)
- Mark C Johnson
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of CambridgeCambridgeUnited Kingdom
| | - Geylani Can
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of CambridgeCambridgeUnited Kingdom
| | - Miguel Monteiro Santos
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of CambridgeCambridgeUnited Kingdom
| | - Diana Alexander
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of CambridgeCambridgeUnited Kingdom
| | - Philip Zegerman
- Wellcome Trust/Cancer Research United Kingdom Gurdon Institute and Department of Biochemistry, University of CambridgeCambridgeUnited Kingdom
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6
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DNA Rereplication Is Susceptible to Nucleotide-Level Mutagenesis. Genetics 2019; 212:445-460. [PMID: 31028114 PMCID: PMC6553831 DOI: 10.1534/genetics.119.302194] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2018] [Accepted: 04/15/2019] [Indexed: 12/12/2022] Open
Abstract
The initiation of eukaryotic DNA replication at replication origins is tightly regulated to prevent re-initiation and re-replication within each cell cycle. This regulation is critical for genome stability as re-replication is an extremely potent inducer... The sources of genome instability, a hallmark of cancer, remain incompletely understood. One potential source is DNA rereplication, which arises when the mechanisms that prevent the reinitiation of replication origins within a single cell cycle are compromised. Using the budding yeast Saccharomyces cerevisiae, we previously showed that DNA rereplication is extremely potent at inducing gross chromosomal alterations and that this arises in part because of the susceptibility of rereplication forks to break. Here, we examine the ability of DNA rereplication to induce nucleotide-level mutations. During normal replication these mutations are restricted by three overlapping error-avoidance mechanisms: the nucleotide selectivity of replicative polymerases, their proofreading activity, and mismatch repair. Using lys2InsEA14, a frameshift reporter that is poorly proofread, we show that rereplication induces up to a 30× higher rate of frameshift mutations and that this mutagenesis is due to passage of the rereplication fork, not secondary to rereplication fork breakage. Rereplication can also induce comparable rates of frameshift and base-substitution mutations in a more general mutagenesis reporter CAN1, when the proofreading activity of DNA polymerase ε is inactivated. Finally, we show that the rereplication-induced mutagenesis of both lys2InsEA14 and CAN1 disappears in the absence of mismatch repair. These results suggest that mismatch repair is attenuated during rereplication, although at most sequences DNA polymerase proofreading provides enough error correction to mitigate the mutagenic consequences. Thus, rereplication can facilitate nucleotide-level mutagenesis in addition to inducing gross chromosomal alterations, broadening its potential role in genome instability.
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7
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Control of Eukaryotic DNA Replication Initiation-Mechanisms to Ensure Smooth Transitions. Genes (Basel) 2019; 10:genes10020099. [PMID: 30700044 PMCID: PMC6409694 DOI: 10.3390/genes10020099] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2018] [Revised: 01/25/2019] [Accepted: 01/25/2019] [Indexed: 02/06/2023] Open
Abstract
DNA replication differs from most other processes in biology in that any error will irreversibly change the nature of the cellular progeny. DNA replication initiation, therefore, is exquisitely controlled. Deregulation of this control can result in over-replication characterized by repeated initiation events at the same replication origin. Over-replication induces DNA damage and causes genomic instability. The principal mechanism counteracting over-replication in eukaryotes is a division of replication initiation into two steps—licensing and firing—which are temporally separated and occur at distinct cell cycle phases. Here, we review this temporal replication control with a specific focus on mechanisms ensuring the faultless transition between licensing and firing phases.
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8
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The Temporal Regulation of S Phase Proteins During G 1. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2018; 1042:335-369. [PMID: 29357066 DOI: 10.1007/978-981-10-6955-0_16] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Successful DNA replication requires intimate coordination with cell-cycle progression. Prior to DNA replication initiation in S phase, a series of essential preparatory events in G1 phase ensures timely, complete, and precise genome duplication. Among the essential molecular processes are regulated transcriptional upregulation of genes that encode replication proteins, appropriate post-transcriptional control of replication factor abundance and activity, and assembly of DNA-loaded protein complexes to license replication origins. In this chapter we describe these critical G1 events necessary for DNA replication and their regulation in the context of both cell-cycle entry and cell-cycle progression.
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9
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Abstract
Replication forks encounter obstacles that must be repaired or bypassed to complete chromosome duplication before cell division. Proteomic analysis of replication forks suggests that the checkpoint and repair machinery travels with unperturbed forks, implying that they are poised to respond to stalling and collapse. However, impaired fork progression still generates aberrations, including repeat copy number instability and chromosome rearrangements. Deregulated origin firing also causes fork instability if a newer fork collides with an older one, generating double-strand breaks (DSBs) and partially rereplicated DNA. Current evidence suggests that multiple mechanisms are used to repair rereplication damage, yet these can have deleterious consequences for genome integrity.
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10
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Abstract
The accurate and complete replication of genomic DNA is essential for all life. In eukaryotic cells, the assembly of the multi-enzyme replisomes that perform replication is divided into stages that occur at distinct phases of the cell cycle. Replicative DNA helicases are loaded around origins of DNA replication exclusively during G1 phase. The loaded helicases are then activated during S phase and associate with the replicative DNA polymerases and other accessory proteins. The function of the resulting replisomes is monitored by checkpoint proteins that protect arrested replisomes and inhibit new initiation when replication is inhibited. The replisome also coordinates nucleosome disassembly, assembly, and the establishment of sister chromatid cohesion. Finally, when two replisomes converge they are disassembled. Studies in Saccharomyces cerevisiae have led the way in our understanding of these processes. Here, we review our increasingly molecular understanding of these events and their regulation.
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Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage. Proc Natl Acad Sci U S A 2016; 114:406-411. [PMID: 28028228 DOI: 10.1073/pnas.1619774114] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Previously, we have shown that loss of the histone 3 lysine 27 (H3K27) monomethyltransferases ARABIDOPSIS TRITHORAX-RELATED 5 (ATXR5) and ATXR6 (ATXR6) results in the overreplication of heterochromatin. Here we show that the overreplication results in DNA damage and extensive chromocenter remodeling into unique structures we have named "overreplication-associated centers" (RACs). RACs have a highly ordered structure with an outer layer of condensed heterochromatin, an inner layer enriched in the histone variant H2AX, and a low-density core containing foci of phosphorylated H2AX (a marker of double-strand breaks) and the DNA-repair enzyme RAD51. atxr5,6 mutants are strongly affected by mutations in DNA repair, such as ATM and ATR. Because of its dense packaging and repetitive DNA sequence, heterochromatin is a challenging environment in which to repair DNA damage. Previous work in animals has shown that heterochromatic breaks are translocated out of the heterochromatic domain for repair. Our results show that atxr5,6 mutants use a variation on this strategy for repairing heterochromatic DNA damage. Rather than being moved to adjacent euchromatic regions, as in animals, heterochromatin undergoes large-scale remodeling to create a compartment with low chromatin density.
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12
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Pozo PN, Cook JG. Regulation and Function of Cdt1; A Key Factor in Cell Proliferation and Genome Stability. Genes (Basel) 2016; 8:genes8010002. [PMID: 28025526 PMCID: PMC5294997 DOI: 10.3390/genes8010002] [Citation(s) in RCA: 74] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2016] [Revised: 12/13/2016] [Accepted: 12/14/2016] [Indexed: 12/30/2022] Open
Abstract
Successful cell proliferation requires efficient and precise genome duplication followed by accurate chromosome segregation. The Cdc10-dependent transcript 1 protein (Cdt1) is required for the first step in DNA replication, and in human cells Cdt1 is also required during mitosis. Tight cell cycle controls over Cdt1 abundance and activity are critical to normal development and genome stability. We review here recent advances in elucidating Cdt1 molecular functions in both origin licensing and kinetochore–microtubule attachment, and we describe the current understanding of human Cdt1 regulation.
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Affiliation(s)
- Pedro N Pozo
- Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
| | - Jeanette Gowen Cook
- Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
- Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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13
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Reusswig KU, Zimmermann F, Galanti L, Pfander B. Robust Replication Control Is Generated by Temporal Gaps between Licensing and Firing Phases and Depends on Degradation of Firing Factor Sld2. Cell Rep 2016; 17:556-569. [DOI: 10.1016/j.celrep.2016.09.013] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 08/10/2016] [Accepted: 09/02/2016] [Indexed: 10/20/2022] Open
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Demchenko Y, Roschke A, Chen WD, Asmann Y, Bergsagel PL, Kuehl WM. Frequent occurrence of large duplications at reciprocal genomic rearrangement breakpoints in multiple myeloma and other tumors. Nucleic Acids Res 2016; 44:8189-98. [PMID: 27353332 PMCID: PMC5041460 DOI: 10.1093/nar/gkw527] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2015] [Accepted: 05/26/2016] [Indexed: 12/11/2022] Open
Abstract
Using a combination of array comparative genomic hybridization, mate pair and cloned sequences, and FISH analyses, we have identified in multiple myeloma cell lines and tumors a novel and recurrent type of genomic rearrangement, i.e. interchromosomal rearrangements (translocations or insertions) and intrachromosomal inversions that contain long (1-4000 kb; median ∼100 kb) identical sequences adjacent to both reciprocal breakpoint junctions. These duplicated sequences were generated from sequences immediately adjacent to the breakpoint from at least one-but sometimes both-chromosomal donor site(s). Tandem duplications had a similar size distribution suggesting the possibility of a shared mechanism for generating duplicated sequences at breakpoints. Although about 25% of apparent secondary rearrangements contained these duplications, primary IGH translocations rarely, if ever, had large duplications at breakpoint junctions. Significantly, these duplications often contain super-enhancers and/or oncogenes (e.g. MYC) that are dysregulated by rearrangements during tumor progression. We also found that long identical sequences often were identified at both reciprocal breakpoint junctions in six of eight other tumor types. Finally, we have been unable to find reports of similar kinds of rearrangements in wild-type or mutant prokaryotes or lower eukaryotes such as yeast.
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Affiliation(s)
- Yulia Demchenko
- Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4265, USA
| | - Anna Roschke
- Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4265, USA
| | - Wei-Dong Chen
- Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4265, USA
| | - Yan Asmann
- Division of Biomedical Statistics and Informatics, Mayo Clinic, 4500 San Pablo Road South, Jacksonville, FL 32224, USA
| | - Peter Leif Bergsagel
- Comprehensive Cancer Center, Mayo Clinic Arizona, 13400 E. Shea Boulevard, Scottsdale, AZ 85259, USA
| | - Walter Michael Kuehl
- Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4265, USA
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High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation. G3-GENES GENOMES GENETICS 2016; 6:993-1012. [PMID: 26865697 PMCID: PMC4825667 DOI: 10.1534/g3.116.027904] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.
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16
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Alexander JL, Barrasa MI, Orr-Weaver TL. Replication fork progression during re-replication requires the DNA damage checkpoint and double-strand break repair. Curr Biol 2015; 25:1654-60. [PMID: 26051888 DOI: 10.1016/j.cub.2015.04.058] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2014] [Revised: 03/02/2015] [Accepted: 04/24/2015] [Indexed: 11/19/2022]
Abstract
Replication origins are under tight regulation to ensure activation occurs only once per cell cycle [1, 2]. Origin re-firing in a single S phase leads to the generation of DNA double-strand breaks (DSBs) and activation of the DNA damage checkpoint [2-7]. If the checkpoint is blocked, cells enter mitosis with partially re-replicated DNA that generates chromosome breaks and fusions [5]. These types of chromosomal aberrations are common in numerous human cancers, suggesting that re-replication events contribute to cancer progression. It was proposed that fork instability and DSBs formed during re-replication are the result of head-to-tail collisions and collapse of adjacent replication forks [3]. However, previously studied systems lack the resolution to determine whether the observed DSBs are generated at sites of fork collisions. Here, we utilize the Drosophila ovarian follicle cells, which exhibit re-replication under precise developmental control [8-10], to model the consequences of re-replication at actively elongating forks. Re-replication occurs from specific replication origins at six genomic loci, termed Drosophila amplicons in follicle cells (DAFCs) [10-12]. Precise developmental timing of DAFC origin firing permits identification of forks at defined points after origin initiation [13, 14]. Here, we show that DAFC re-replication causes fork instability and generates DSBs at sites of potential fork collisions. Immunofluorescence and ChIP-seq demonstrate the DSB marker γH2Av is enriched at elongating forks. Fork progression is reduced in the absence of DNA damage checkpoint components and nonhomologous end-joining (NHEJ), but not homologous recombination. NHEJ appears to continually repair forks during re-replication to maintain elongation.
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Affiliation(s)
- Jessica L Alexander
- Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 68-132, Cambridge, MA 02139, USA
| | - M Inmaculada Barrasa
- Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA
| | - Terry L Orr-Weaver
- Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 68-132, Cambridge, MA 02139, USA.
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17
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Re-replication of a centromere induces chromosomal instability and aneuploidy. PLoS Genet 2015; 11:e1005039. [PMID: 25901968 PMCID: PMC4406714 DOI: 10.1371/journal.pgen.1005039] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2014] [Accepted: 01/28/2015] [Indexed: 12/19/2022] Open
Abstract
The faithful inheritance of chromosomes during cell division requires their precise replication and segregation. Numerous mechanisms ensure that each of these fundamental cell cycle events is performed with a high degree of fidelity. The fidelity of chromosomal replication is maintained in part by re-replication controls that ensure there are no more than two copies of every genomic segment to distribute to the two daughter cells. This control is enforced by inhibiting replication initiation proteins from reinitiating replication origins within a single cell cycle. Here we show in Saccharomyces cerevisiae that re-replication control is important for the fidelity of chromosome segregation. In particular, we demonstrate that transient re-replication of centromeric DNA due to disruption of re-replication control greatly induces aneuploidy of the re-replicated chromosome. Some of this aneuploidy arises from missegregation of both sister chromatids to one daughter cell. Aneuploidy can also arise from the generation of an extra sister chromatid via homologous recombination, suggesting that centromeric re-replication can trigger breakage and repair events that expand chromosome number without causing chromosomal rearrangements. Thus, we have identified a potential new non-mitotic source of aneuploidy that can arise from a defect in re-replication control. Given the emerging connections between the deregulation of replication initiation proteins and oncogenesis, this finding may be relevant to the aneuploidy that is prevalent in cancer. The stable inheritance of genetic information requires an elaborate mitotic machinery that acts on the centromeres of chromosomes to ensure their precise segregation. Errors in this segregation can lead to aneuploidy, an unbalanced chromosomal state in which some chromosomes have different copy number than others. Because aneuploidy is associated with developmental abnormalities and diseases such as cancer, there is considerable interest in understanding how these segregation errors arise. Much of this interest has focused on identifying defects in proteins that make up the mitotic machinery. Here, we show that defects in a completely separate process, the control of DNA replication initiation, can lead to chromosome segregation errors as a result of inappropriate re-replication of centromeres. Similar deregulation of replication initiation proteins has been observed in primary human tumors and shown to promote oncogenesis in mouse models. Together, these results raise the possibility that centromeric re-replication may be an additional source of aneuploidy in cancer. In combination with our previous work showing that re-replication is a potent inducer of gene amplification, these results also highlight the versatility of re-replication as a source of genomic instability.
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18
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Zhang J, Zuo T, Wang D, Peterson T. Transposition-mediated DNA re-replication in maize. eLife 2014; 3:e03724. [PMID: 25406063 PMCID: PMC4270019 DOI: 10.7554/elife.03724] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2014] [Accepted: 11/17/2014] [Indexed: 02/03/2023] Open
Abstract
Every DNA segment in a eukaryotic genome normally replicates once and only once per cell cycle to maintain genome stability. We show here that this restriction can be bypassed through alternative transposition, a transposition reaction that utilizes the termini of two separate, nearby transposable elements (TEs). Our results suggest that alternative transposition during S phase can induce re-replication of the TEs and their flanking sequences. The DNA re-replication can spontaneously abort to generate double-strand breaks, which can be repaired to generate Composite Insertions composed of transposon termini flanking segmental duplications of various lengths. These results show how alternative transposition coupled with DNA replication and repair can significantly alter genome structure and may have contributed to rapid genome evolution in maize and possibly other eukaryotes.
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Affiliation(s)
- Jianbo Zhang
- Department of Genetics,
Development and Cell Biology, Iowa State
University, Ames, United States
- Department of
Agronomy, Iowa State University,
Ames,
United States
| | - Tao Zuo
- Department of Genetics,
Development and Cell Biology, Iowa State
University, Ames, United States
- Department of
Agronomy, Iowa State University,
Ames,
United States
| | - Dafang Wang
- Department of Genetics,
Development and Cell Biology, Iowa State
University, Ames, United States
- Department of
Agronomy, Iowa State University,
Ames,
United States
| | - Thomas Peterson
- Department of Genetics,
Development and Cell Biology, Iowa State
University, Ames, United States
- Department of
Agronomy, Iowa State University,
Ames,
United States
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19
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Chao TC, Chen KJ, Tang MC, Chan LC, Chen PM, Tzeng CH, Su Y. Thymosin beta-4 knockdown in IEC-6 normal intestinal epithelial cells induces DNA re-replication via downregulating Emi1. J Cell Physiol 2014; 229:1639-46. [PMID: 24615569 DOI: 10.1002/jcp.24609] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2014] [Accepted: 03/04/2014] [Indexed: 12/19/2022]
Abstract
Thymosin β4 (Tβ4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tβ4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3β (GSK-3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4 .
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Affiliation(s)
- Ta-Chung Chao
- Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan
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20
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Truong LN, Li Y, Sun E, Ang K, Hwang PYH, Wu X. Homologous recombination is a primary pathway to repair DNA double-strand breaks generated during DNA rereplication. J Biol Chem 2014; 289:28910-23. [PMID: 25160628 DOI: 10.1074/jbc.m114.576488] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Re-initiation of DNA replication at origins within a given cell cycle would result in DNA rereplication, which can lead to genome instability and tumorigenesis. DNA rereplication can be induced by loss of licensing control at cellular replication origins, or by viral protein-driven multiple rounds of replication initiation at viral origins. DNA double-strand breaks (DSBs) are generated during rereplication, but the mechanisms of how these DSBs are repaired to maintain genome stability and cell viability are poorly understood in mammalian cells. We generated novel EGFP-based DSB repair substrates, which specifically monitor the repair of rereplication-associated DSBs. We demonstrated that homologous recombination (HR) is an important mechanism to repair rereplication-associated DSBs, and sister chromatids are used as templates for such HR-mediated DSB repair. Micro-homology-mediated non-homologous end joining (MMEJ) can also be used but to a lesser extent compared to HR, whereas Ku-dependent classical non-homologous end joining (C-NHEJ) has a minimal role to repair rereplication-associated DSBs. In addition, loss of HR activity leads to severe cell death when rereplication is induced. Therefore, our studies identify HR, the most conservative repair pathway, as the primary mechanism to repair DSBs upon rereplication.
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Affiliation(s)
- Lan N Truong
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
| | - Yongjiang Li
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
| | - Emily Sun
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
| | - Katrina Ang
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
| | - Patty Yi-Hwa Hwang
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
| | - Xiaohua Wu
- From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037
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21
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Abstract
DNA replication must be tightly regulated to ensure that the genome is accurately duplicated during each cell cycle. When these regulatory mechanisms fail, replicative stress and DNA damage ensue. Activated oncogenes promote replicative stress, inducing a DNA damage response (DDR) early in tumorigenesis. Senescence or apoptosis result, forming a barrier against tumour progression. This may provide a selective pressure for acquisition of mutations in the DDR pathway during tumorigenesis. Despite its potential importance in early cancer development, the precise nature of oncogene-induced replicative stress remains poorly understood. Here, we review our current understanding of replication initiation and its regulation, describe mechanisms by which activated oncogenes might interfere with these processes and discuss how replicative stress might contribute to the genomic instability seen in cancers.
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Affiliation(s)
- Stephanie A Hills
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, UK
| | - John F X Diffley
- Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, UK.
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22
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Single-stranded annealing induced by re-initiation of replication origins provides a novel and efficient mechanism for generating copy number expansion via non-allelic homologous recombination. PLoS Genet 2013; 9:e1003192. [PMID: 23300490 PMCID: PMC3536649 DOI: 10.1371/journal.pgen.1003192] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2012] [Accepted: 11/08/2012] [Indexed: 11/24/2022] Open
Abstract
Copy number expansions such as amplifications and duplications contribute to human phenotypic variation, promote molecular diversification during evolution, and drive the initiation and/or progression of various cancers. The mechanisms underlying these copy number changes are still incompletely understood, however. We recently demonstrated that transient, limited re-replication from a single origin in Saccharomyces cerevisiae efficiently induces segmental amplification of the re-replicated region. Structural analyses of such re-replication induced gene amplifications (RRIGA) suggested that RRIGA could provide a new mechanism for generating copy number variation by non-allelic homologous recombination (NAHR). Here we elucidate this new mechanism and provide insight into why it is so efficient. We establish that sequence homology is both necessary and sufficient for repetitive elements to participate in RRIGA and show that their recombination occurs by a single-strand annealing (SSA) mechanism. We also find that re-replication forks are prone to breakage, accounting for the widespread DNA damage associated with deregulation of replication proteins. These breaks appear to stimulate NAHR between re-replicated repeat sequences flanking a re-initiating replication origin. Our results support a RRIGA model where the expansion of a re-replication bubble beyond flanking homologous sequences followed by breakage at both forks in trans provides an ideal structural context for SSA–mediated NAHR to form a head-to-tail duplication. Given the remarkable efficiency of RRIGA, we suggest it may be an unappreciated contributor to copy number expansions in both disease and evolution. Duplications and amplifications of chromosomal segments are frequently observed in eukaryotic genomes, including both normal and cancerous human genomes. These copy number variations contribute to the phenotypic variation upon which natural selection acts. For example, the amplification of genes whose excessive copy number facilitates uncontrolled cell division is often selected for during tumor development. Copy number variations can often arise when repetitive sequence elements, which are dispersed throughout eukaryotic genomes, undergo a rearrangement called non-allelic homologous recombination. Exactly how these rearrangements occur is poorly understood. Here, using budding yeast to model this class of copy number variation, we uncover a new and highly efficient mechanism by which these variations can be generated. The precipitating event is the aberrant re-initiation of DNA replication at a replication origin. Normally the hundreds to thousands of origins scattered throughout a eukaryotic genome are tightly controlled such that each is permitted to initiate only once per cell cycle. However, disruptions in these controls can allow origins to re-initiate, and we show how the resulting DNA re-replication structure can be readily converted into a tandem duplication via non-allelic homologous recombination. Hence, the re-initiation of DNA replication is a potential source of copy number variation both in disease and during evolution.
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23
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Klotz-Noack K, McIntosh D, Schurch N, Pratt N, Blow JJ. Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation. J Cell Sci 2012; 125:2436-45. [PMID: 22366459 PMCID: PMC3481538 DOI: 10.1242/jcs.100883] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.
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Affiliation(s)
- Kathleen Klotz-Noack
- Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
| | - Debbie McIntosh
- Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
| | - Nicholas Schurch
- Data Analysis Group, College of Life Sciences, University of Dundee DD1 5EH, UK
| | - Norman Pratt
- Department of Human Genetics, Ninewells Hospital, Dundee DD1 9SY, UK
| | - J. Julian Blow
- Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
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24
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Tanaka S, Araki H. Multiple regulatory mechanisms to inhibit untimely initiation of DNA replication are important for stable genome maintenance. PLoS Genet 2011; 7:e1002136. [PMID: 21698130 PMCID: PMC3116906 DOI: 10.1371/journal.pgen.1002136] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2010] [Accepted: 05/01/2011] [Indexed: 12/28/2022] Open
Abstract
Genomic instability is a hallmark of human cancer cells. To prevent genomic instability, chromosomal DNA is faithfully duplicated in every cell division cycle, and eukaryotic cells have complex regulatory mechanisms to achieve this goal. Here, we show that untimely activation of replication origins during the G1 phase is genotoxic and induces genomic instability in the budding yeast Saccharomyces cerevisiae. Our data indicate that cells preserve a low level of the initiation factor Sld2 to prevent untimely initiation during the normal cell cycle in addition to controlling the phosphorylation of Sld2 and Sld3 by cyclin-dependent kinase. Although untimely activation of origin is inhibited on multiple levels, we show that deregulation of a single pathway can cause genomic instability, such as gross chromosome rearrangements (GCRs). Furthermore, simultaneous deregulation of multiple pathways causes an even more severe phenotype. These findings highlight the importance of having multiple inhibitory mechanisms to prevent the untimely initiation of chromosome replication to preserve stable genome maintenance over generations in eukaryotes. Chromosomal DNA replication occurs as a two-step reaction in eukaryotes. In the first reaction, called licensing, the replicative helicase is loaded onto replication origin in an inactive form during the G1 phase of the cell cycle. In the second reaction, called initiation, the replicative helicase is activated, and replication forks are established. Because of this two-step mechanism, licensing and initiation must occur at different times in the cell cycle. Failure of this two-step regulation will cause heterogeneous re-replication of chromosomal DNA, and genome integrity will be lost. Although previous works have established that multiple regulatory pathways regulate licensing, much less is known about how untimely (premature) initiation is prevented during the G1 phase. In this paper, we show that untimely activation of replication origins during the G1 phase is inhibited on multiple levels. Notably, deregulation of a single pathway can cause genomic instability; simultaneous deregulation of multiple pathways causes a more severe phenotype, such as aneuploidy. Therefore, these findings not only indicate the importance of having multiple inhibitory mechanisms to prevent untimely initiation of chromosome replication but also should help us understand how replication might be deregulated in human cancer cells, in which the genome is frequently destabilized.
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Affiliation(s)
- Seiji Tanaka
- Division of Microbial Genetics, National Institute of Genetics, Mishima, Japan.
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25
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Milhollen MA, Narayanan U, Soucy TA, Veiby PO, Smith PG, Amidon B. Inhibition of NEDD8-activating enzyme induces rereplication and apoptosis in human tumor cells consistent with deregulating CDT1 turnover. Cancer Res 2011; 71:3042-51. [PMID: 21487042 DOI: 10.1158/0008-5472.can-10-2122] [Citation(s) in RCA: 132] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.
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Affiliation(s)
- Michael A Milhollen
- Discovery, Millennium Pharmaceuticals, Inc, Cambridge, Massachusetts 02139, USA.
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26
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Ding Q, MacAlpine DM. Preferential re-replication of Drosophila heterochromatin in the absence of geminin. PLoS Genet 2010; 6:e1001112. [PMID: 20838463 PMCID: PMC2936543 DOI: 10.1371/journal.pgen.1001112] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2010] [Accepted: 08/05/2010] [Indexed: 01/04/2023] Open
Abstract
To ensure genomic integrity, the genome must be duplicated exactly once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. Cdt1, also known as Double-parked (Dup) in Drosophila, is a key regulator of the assembly of the pre-replicative complex (pre-RC) and its activity is strictly limited to G1 by multiple mechanisms including Cul4-Ddb1 mediated proteolysis and inhibition by geminin. We assayed the genomic consequences of disregulating the replication licensing mechanisms by RNAi depletion of geminin. We found that not all origins of replication were sensitive to geminin depletion and that heterochromatic sequences were preferentially re-replicated in the absence of licensing mechanisms. The preferential re-activation of heterochromatic origins of replication was unexpected because these are typically the last sequences to be duplicated in a normal cell cycle. We found that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather by the formation of the pre-RC. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin by elevated cyclin A-CDK activity. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1. Catastrophic consequences may occur if the cell fails to either completely copy the genome or if it duplicates some regions of the genome more than once in a cell cycle. The cell must coordinate thousands of DNA replication start sites (origins) to ensure that the entire genome is copied and that no replication origin is activated more than once in a cell cycle. The cell accomplishes this coordination by confining the selection and activation of replication origins to discrete phases of the cell cycle. Start sites can only be selected or ‘licensed’ for DNA replication in G1 and similarly, they can only be activated for the initiation of DNA replication in S phase. Disruption of the mechanisms that regulate this ‘licensing’ process have been shown to result in extensive re-replication, genomic instability and tumorigenesis in a variety of eukaryotic systems. Here we use genomic approaches in Drosophila to identify which origins of replication are susceptible to re-initiation of DNA replication in the absence of replication licensing controls. Unexpectedly, we find that sequences in the heterochromatin, which were thought to contain only inefficient origins of replication, are preferentially re-replicated. These results provide insights into how origins of replication are selected and regulated in distinct chromatin environments to maintain genomic stability.
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Affiliation(s)
- Queying Ding
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America
| | - David M. MacAlpine
- Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, United States of America
- * E-mail:
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27
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Green BM, Finn KJ, Li JJ. Loss of DNA replication control is a potent inducer of gene amplification. Science 2010; 329:943-6. [PMID: 20724634 DOI: 10.1126/science.1190966] [Citation(s) in RCA: 99] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is inappropriately re-replicated, but the importance of this stringent control on genome stability has not been tested. Here we show that re-replication in Saccharomyces cerevisiae can strongly induce the initial step of gene amplification, increasing gene copy number from one to two or more. The resulting amplicons consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. These re-replication-induced gene amplifications are mediated by nonallelic homologous recombination between the repetitive elements. We suggest that re-replication may be a contributor to gene copy number changes, which are important in fields such as cancer biology, evolution, and human genetics.
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Affiliation(s)
- Brian M Green
- Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
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28
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Regulation of the initiation step of DNA replication by cyclin-dependent kinases. Chromosoma 2010; 119:565-74. [DOI: 10.1007/s00412-010-0291-8] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2010] [Revised: 07/23/2010] [Accepted: 07/23/2010] [Indexed: 12/20/2022]
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29
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Cook JG. Replication licensing and the DNA damage checkpoint. Front Biosci (Landmark Ed) 2009; 14:5013-30. [PMID: 19482602 DOI: 10.2741/3584] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings suggest that checkpoints and replication are more intimately associated than previously appreciated, even in the absence of exogenous DNA damage. This review summarizes some of these developments.
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Affiliation(s)
- Jeanette Gowen Cook
- Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center Campus Box 7260, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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30
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Alberghina L, Coccetti P, Orlandi I. Systems biology of the cell cycle of Saccharomyces cerevisiae: From network mining to system-level properties. Biotechnol Adv 2009; 27:960-978. [PMID: 19465107 DOI: 10.1016/j.biotechadv.2009.05.021] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Following a brief description of the operational procedures of systems biology (SB), the cell cycle of budding yeast is discussed as a successful example of a top-down SB analysis. After the reconstruction of the steps that have led to the identification of a sizer plus timer network in the G1 to S transition, it is shown that basic functions of the cell cycle (the setting of the critical cell size and the accuracy of DNA replication) are system-level properties, detected only by integrating molecular analysis with modelling and simulation of their underlying networks. A detailed network structure of a second relevant regulatory step of the cell cycle, the exit from mitosis, derived from extensive data mining, is constructed and discussed. To reach a quantitative understanding of how nutrients control, through signalling, metabolism and transcription, cell growth and cycle is a very relevant aim of SB. Since we know that about 900 gene products are required for cell cycle execution and control in budding yeast, it is quite clear that a purely systematic approach would require too much time. Therefore lines for a modular SB approach, which prioritises molecular and computational investigations for faster cell cycle understanding, are proposed. The relevance of the insight coming from the cell cycle SB studies in developing a new framework for tackling very complex biological processes, such as cancer and aging, is discussed.
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Affiliation(s)
- Lilia Alberghina
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milano, Italy.
| | - Paola Coccetti
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milano, Italy
| | - Ivan Orlandi
- Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milano, Italy
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Mehrotra S, Maqbool SB, Kolpakas A, Murnen K, Calvi BR. Endocycling cells do not apoptose in response to DNA rereplication genotoxic stress. Genes Dev 2009; 22:3158-71. [PMID: 19056894 DOI: 10.1101/gad.1710208] [Citation(s) in RCA: 114] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Initiation of DNA replication at origins more than once per cell cycle results in rereplication and has been implicated in cancer. Here we use Drosophila to examine the checkpoint responses to rereplication in a developmental context. We find that increased Double-parked (Dup), the Drosophila ortholog of Cdt1, results in rereplication and DNA damage. In most cells, this rereplication triggers caspase activation and apoptotic cell death mediated by both p53-dependent and -independent pathways. Elevated Dup also caused DNA damage in endocycling cells, which switch to a G/S cycle during normal development, indicating that rereplication and the endocycling DNA reduplication program are distinct processes. Unexpectedly, however, endocycling cells do not apoptose regardless of tissue type. Our combined evidence suggests that endocycling apoptosis is repressed in part because proapoptotic gene promoters are silenced. Normal endocycling cells had DNA lesions near heterochromatin, which increased after rereplication, explaining why endocycling cells must constantly repress the genotoxic apoptotic response. Our results reveal a novel regulation of apoptosis in development and new insights into the little-understood endocycle. Similar mechanisms may operate during vertebrate development, with implications for cancer predisposition in certain tissues.
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Affiliation(s)
- Sonam Mehrotra
- Department of Biology, Syracuse University, Syracuse, New York 13244, USA
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Abstract
Correct regulation of the replication licensing system ensures that chromosomal DNA is precisely duplicated in each cell division cycle. Licensing proteins are inappropriately expressed at an early stage of tumorigenesis in a wide variety of cancers. Here we discuss evidence that misregulation of replication licensing is a consequence of oncogene-induced cell proliferation. This misregulation can cause either under- or over-replication of chromosomal DNA, and could explain the genetic instability commonly seen in cancer cells.
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Affiliation(s)
- J Julian Blow
- Wellcome Trust Centre for Gene Regulation & Expression, University of Dundee, DD1 5EH, UK.
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Mickle KL, Oliva A, Huberman JA, Leatherwood J. Checkpoint effects and telomere amplification during DNA re-replication in fission yeast. BMC Mol Biol 2007; 8:119. [PMID: 18154680 PMCID: PMC2265721 DOI: 10.1186/1471-2199-8-119] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2007] [Accepted: 12/21/2007] [Indexed: 11/26/2022] Open
Abstract
Background Although much is known about molecular mechanisms that prevent re-initiation of DNA replication on newly replicated DNA during a single cell cycle, knowledge is sparse regarding the regions that are most susceptible to re-replication when those mechanisms are bypassed and regarding the extents to which checkpoint pathways modulate re-replication. We used microarrays to learn more about these issues in wild-type and checkpoint-mutant cells of the fission yeast, Schizosaccharomyces pombe. Results We found that over-expressing a non-phosphorylatable form of the replication-initiation protein, Cdc18 (known as Cdc6 in other eukaryotes), drove re-replication of DNA sequences genome-wide, rather than forcing high level amplification of just a few sequences. Moderate variations in extents of re-replication generated regions spanning hundreds of kilobases that were amplified (or not) ~2-fold more (or less) than average. However, these regions showed little correlation with replication origins used during S phase. The extents and locations of amplified regions in cells deleted for the checkpoint genes encoding Rad3 (ortholog of human ATR and budding yeast Mec1) and Cds1 (ortholog of human Chk2 and budding yeast Rad53) were similar to those in wild-type cells. Relatively minor but distinct effects, including increased re-replication of heterochromatic regions, were found specifically in cells lacking Rad3. These might be due to Cds1-independent roles for Rad3 in regulating re-replication and/or due to the fact that cells lacking Rad3 continued to divide during re-replication, unlike wild-type cells or cells lacking Cds1. In both wild-type and checkpoint-mutant cells, regions near telomeres were particularly susceptible to re-replication. Highly re-replicated telomere-proximal regions (50–100 kb) were, in each case, followed by some of the least re-replicated DNA in the genome. Conclusion The origins used, and the extent of replication fork progression, during re-replication are largely independent of the replication and DNA-damage checkpoint pathways mediated by Cds1 and Rad3. The fission yeast pattern of telomere-proximal amplification adjacent to a region of under-replication has also been seen in the distantly-related budding yeast, which suggests that subtelomeric sequences may be a promising place to look for DNA re-replication in other organisms.
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Affiliation(s)
- Katie L Mickle
- Department of Microbiology and Molecular Genetics, SUNY at Stony Brook, Stony Brook, New York 11794-5222, USA.
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Liu E, Lee AYL, Chiba T, Olson E, Sun P, Wu X. The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted. ACTA ACUST UNITED AC 2007; 179:643-57. [PMID: 18025301 PMCID: PMC2080923 DOI: 10.1083/jcb.200704138] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
Abstract
DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.
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Affiliation(s)
- Enbo Liu
- Department of Molecular Experimental Medicine and 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
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Lee AYL, Liu E, Wu X. The Mre11/Rad50/Nbs1 complex plays an important role in the prevention of DNA rereplication in mammalian cells. J Biol Chem 2007; 282:32243-55. [PMID: 17715134 DOI: 10.1074/jbc.m705486200] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Mre11/Nbs1/Rad50 complex (MRN) plays multiple roles in the maintenance of genome stability, including repair of double-stranded breaks (DSBs) and activation of the S-phase checkpoint. Here we demonstrate that MRN is required for the prevention of DNA rereplication in mammalian cells. DNA replication is strictly regulated by licensing control so that the genome is replicated once and only once per cell cycle. Inactivation of Nbs1 or Mre11 leads to a substantial increase of DNA rereplication induced by overexpression of the licensing factor Cdt1. Our studies reveal that multiple mechanisms are likely involved in the MRN-mediated suppression of rereplication. First, both Mre11 and Nbs1 are required for facilitating ATR activation when Cdt1 is overexpressed, which in turn suppresses rereplication. Second, Cdt1 overexpression induces ATR-mediated phosphorylation of Nbs1 at Ser343 and this phosphorylation depends on the FHA and BRCT domains of Nbs1. Mutations at Ser343 or in the FHA and BRCT domains lead to more severe rereplication when Cdt1 is overexpressed. Third, the interaction of the Mre11 complex with RPA is important for the suppression of rereplication. This suggests that modulating RPA activity via a direct interaction of MRN is likely one of the effector mechanisms to suppress rereplication. Moreover, we demonstrate that MRN is also required for preventing the accumulation of DSBs when rereplication is induced. Therefore, our studies suggest new roles of MRN in the maintenance of genome stability through preventing rereplication and rereplication-associated DSBs when licensing control is compromised.
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Affiliation(s)
- Alan Yueh-Luen Lee
- Department of Molecular Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA
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36
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Conti C, Herrick J, Bensimon A. Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon. Genes Chromosomes Cancer 2007; 46:724-34. [PMID: 17444495 DOI: 10.1002/gcc.20448] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene.
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Affiliation(s)
- Chiara Conti
- Genomic Vision, 27 rue du Faubourg Saint Jacques, 75014 Paris, France
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37
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Klassen R, Krampe S, Meinhardt F. Homologous recombination and the yKu70/80 complex exert opposite roles in resistance against the killer toxin from Pichia acaciae. DNA Repair (Amst) 2007; 6:1864-75. [PMID: 17765020 DOI: 10.1016/j.dnarep.2007.07.010] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2007] [Revised: 06/25/2007] [Accepted: 07/12/2007] [Indexed: 12/28/2022]
Abstract
The linear plasmid (pPac1-2) encoded killer toxin (PaT) of the yeast Pichia acaciae arrests sensitive Saccharomyces cerevisiae cells in the S-phase of the cell cycle and induces mutations. Here we provide evidence for opposite effects in PaT resistance of homologous recombination (HR) and non-homologous end joining (NHEJ), the two alternative repair mechanisms acting on DNA double strand breaks (DSB). As mutants defective in genes of the RAD52 epistasis group react hypersensitive and cells lacking YKU70 or YKU80 are partially resistant, the yKu70/80 complex facilitates PaT toxicity, whereas HR is antagonistic. In contrast to yku70 and yku80, lif1 mutants, the latter being defective in the ligation step of NHEJ, are PaT sensitive, confining toxicity promoting effects of NHEJ to the DSB end binding Ku proteins. Since rad52 yku80 double mutants display strong hypersensitivity, yku80 mediated resistance depends on HR. Opposite effects of the yKu70/80 complex and HR are consistent with the occurrence of replication dependent (one sided) DSBs in PaT treated cells. Concordantly, two cellular markers signaling DSBs are induced during PaT mediated S-phase arrest, i.e. histone H2A phosphorylation and formation of subnuclear repair foci by GFP tagged recombination protein Rad52. As only moderate chromosome fragmentation could be detected by PFGE, transient occurrence and efficient in vivo repair of PaT induced DSBs is assumed. Consistent with replication dependent DSB formation induced by PaT, we demonstrate a protective function of the RecQ helicase Sgs1 and the structure specific endonuclease Mus81, both of which are considered to be involved in processing and restart of stalled replication forks.
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Affiliation(s)
- Roland Klassen
- Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, D-48149 Münster, Germany
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38
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Arias EE, Walter JC. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev 2007; 21:497-518. [PMID: 17344412 DOI: 10.1101/gad.1508907] [Citation(s) in RCA: 313] [Impact Index Per Article: 17.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
In eukaryotic cells, prereplication complexes (pre-RCs) are assembled on chromatin in the G1 phase, rendering origins of DNA replication competent to initiate DNA synthesis. When DNA replication commences in S phase, pre-RCs are disassembled, and multiple initiations from the same origin do not occur because new rounds of pre-RC assembly are inhibited. In most experimental organisms, multiple mechanisms that prevent pre-RC assembly have now been identified, and rereplication within the same cell cycle can be induced through defined perturbations of these mechanisms. This review summarizes the diverse array of inhibitory pathways used by different organisms to prevent pre-RC assembly, and focuses on the challenge of understanding how in any one cell type, various mechanisms cooperate to strictly enforce once per cell cycle regulation of DNA replication.
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Affiliation(s)
- Emily E Arias
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA
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39
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Honey S, Futcher B. Roles of the CDK phosphorylation sites of yeast Cdc6 in chromatin binding and rereplication. Mol Biol Cell 2007; 18:1324-36. [PMID: 17267692 PMCID: PMC1838967 DOI: 10.1091/mbc.e06-06-0544] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.
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Affiliation(s)
- Sangeet Honey
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222
| | - Bruce Futcher
- Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794-5222
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40
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Ikui AE, Archambault V, Drapkin BJ, Campbell V, Cross FR. Cyclin and cyclin-dependent kinase substrate requirements for preventing rereplication reveal the need for concomitant activation and inhibition. Genetics 2006; 175:1011-22. [PMID: 17194775 PMCID: PMC1840059 DOI: 10.1534/genetics.106.068213] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
DNA replication initiation in S. cerevisiae is promoted by B-type cyclin-dependent kinase (Cdk) activity. In addition, once-per-cell-cycle replication is enforced by cyclin-Cdk-dependent phosphorylation of the prereplicative complex (pre-RC) components Mcm2-7, Cdc6, and Orc1-6. Several of these controls must be simultaneously blocked by mutation to obtain rereplication. We looked for but did not obtain strong evidence for cyclin specificity in the use of different mechanisms to control rereplication: both the S-phase cyclin Clb5 and the mitotic cyclins Clb1-4 were inferred to be capable of imposing ORC-based and MCM-based controls. We found evidence that the S-phase cyclin Clb6 could promote initiation of replication without blocking reinitiation, and this activity was highly toxic when the ability of other cyclins to block reinitiation was prevented by mutation. The failure of Clb6 to regulate reinitiation was due to rapid Clb6 proteolysis, since this toxic activity of Clb6 was lost when Clb6 was stabilized by mutation. Clb6-dependent toxicity is also relieved when early accumulation of mitotic cyclins is allowed to impose rereplication controls. Cell-cycle timing of rereplication control is crucial: sufficient rereplication block activity must be available as soon as firing begins. DNA rereplication induces DNA damage, and when rereplication controls are compromised, the DNA damage checkpoint factors Mre11 and Rad17 provide additional mechanisms that maintain viability and also prevent further rereplication, and this probably contributes to genome stability.
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Affiliation(s)
- Amy E Ikui
- The Rockefeller University, New York, New York 10021, USA
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41
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Davidson IF, Li A, Blow JJ. Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision. Mol Cell 2006; 24:433-43. [PMID: 17081992 PMCID: PMC1819398 DOI: 10.1016/j.molcel.2006.09.010] [Citation(s) in RCA: 113] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2006] [Revised: 08/07/2006] [Accepted: 09/18/2006] [Indexed: 12/29/2022]
Abstract
Correct regulation of the replication licensing system ensures that no DNA is rereplicated in a single cell cycle. When the licensing protein Cdt1 is overexpressed in G2 phase of the cell cycle, replication origins are relicensed and the DNA is rereplicated. At the same time, checkpoint pathways are activated that block further cell cycle progression. We have studied the consequence of deregulating the licensing system by adding recombinant Cdt1 to Xenopus egg extracts. We show that Cdt1 induces checkpoint activation and the appearance of small fragments of double-stranded DNA. DNA fragmentation and strong checkpoint activation are dependent on uncontrolled rereplication and do not occur after a single coordinated round of rereplication. The DNA fragments are composed exclusively of rereplicated DNA. The unusual characteristics of these fragments suggest that they result from head-to-tail collision (rear ending) of replication forks chasing one another along the same DNA template.
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Affiliation(s)
- Iain F. Davidson
- School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
| | - Anatoliy Li
- School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
| | - J. Julian Blow
- School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom
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42
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Laha S, Das SP, Hajra S, Sau S, Sinha P. The budding yeast protein Chl1p is required to preserve genome integrity upon DNA damage in S-phase. Nucleic Acids Res 2006; 34:5880-91. [PMID: 17062629 PMCID: PMC1635322 DOI: 10.1093/nar/gkl749] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The budding yeast protein, Chl1p, is required for sister-chromatid cohesion, transcriptional silencing, rDNA recombination and aging. In this work, we show that Chl1p is also required for viability when DNA replication is stressed, either due to mutations or if cells are treated with genotoxic agents like methylmethane sulfonate (MMS) and ultraviolet (UV) rays. The chl1 mutation caused synthetic growth defects with mutations in DNA replication genes. At semi-permissive temperatures, the double mutants grew poorly, were less viable and showed nuclear fragmentation. They were, however, not limited in their bulk DNA synthesis. When chl1 cells were treated with relatively low levels of MMS in S-phase, they lost viability. The S-phase DNA damage checkpoint pathway, however, remained active in these cells. Agarose gel electrophoresis of genomic DNA isolated from wild-type and chl1 cells, after recovery from MMS treatment, suggested that the wild-type was more proficient in the repair of DNA damage than the mutant. Our work suggests that Chl1p is required for genome integrity when cells suffer endogenously or exogenously induced DNA damage.
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Affiliation(s)
| | | | | | | | - Pratima Sinha
- To whom correspondence should be addressed. Tel: 91 33 23550256; Fax: 91 33 23343886;
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43
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Lovejoy CA, Lock K, Yenamandra A, Cortez D. DDB1 maintains genome integrity through regulation of Cdt1. Mol Cell Biol 2006; 26:7977-90. [PMID: 16940174 PMCID: PMC1636754 DOI: 10.1128/mcb.00819-06] [Citation(s) in RCA: 76] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.
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Affiliation(s)
- Courtney A Lovejoy
- Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, USA
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44
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Tatsumi Y, Sugimoto N, Yugawa T, Narisawa-Saito M, Kiyono T, Fujita M. Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability. J Cell Sci 2006; 119:3128-40. [PMID: 16835273 DOI: 10.1242/jcs.03031] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The activity of human Cdt1 is negatively regulated by multiple mechanisms. This suggests that Cdt1 deregulation may have a deleterious effect. Indeed, it has been suggested that overexpression of Cdt1 can induce rereplication in cancer cells and that rereplication activates Ataxia-telangiectasia-mutated (ATM) kinase and/or ATM- and Rad3-related (ATR) kinase-dependent checkpoint pathways. In this report, we highlight a new and interesting aspect of Cdt1 deregulation: data from several different systems all strongly indicate that unregulated Cdt1 overexpression at pathophysiological levels can induce chromosomal damage other than rereplication in non-transformed cells. The most important finding in these studies is that deregulated Cdt1 induces chromosomal damage and activation of the ATM-Chk2 DNA damage checkpoint pathway even in quiescent cells. These Cdt1 activities are negatively regulated by cyclin A/Cdks, probably through modification by phosphorylation. Furthermore, we found that deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this would be a new molecular mechanism leading to carcinogenesis.
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Affiliation(s)
- Yasutoshi Tatsumi
- Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045, Japan
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45
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Celic I, Masumoto H, Griffith WP, Meluh P, Cotter RJ, Boeke JD, Verreault A. The Sirtuins Hst3 and Hst4p Preserve Genome Integrity by Controlling Histone H3 Lysine 56 Deacetylation. Curr Biol 2006; 16:1280-9. [PMID: 16815704 DOI: 10.1016/j.cub.2006.06.023] [Citation(s) in RCA: 238] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2006] [Revised: 05/06/2006] [Accepted: 06/07/2006] [Indexed: 11/20/2022]
Abstract
BACKGROUND Acetylation of histone H3 lysine 56 (K56Ac) occurs transiently in newly synthesized H3 during passage through S phase and is removed in G2. However, the physiologic roles and effectors of K56Ac turnover are unknown. RESULTS The sirtuins Hst3p and, to a lesser extent, Hst4p maintain low levels of K56Ac outside of S phase. In hst3 hst4 mutants, K56 hyperacetylation nears 100%. Residues corresponding to the nicotinamide binding pocket of Sir2p are essential for Hst3p function, and H3 K56 deacetylation is inhibited by nicotinamide in vivo. Rapid inactivation of Hst3/Hst4p prior to S phase elevates K56Ac to 50% in G2, suggesting that K56-acetylated nucleosomes are assembled genome-wide during replication. Inducible expression of Hst3p in G1 or G2 triggers deacetylation of mature chromatin. Cells lacking Hst3/Hst4p exhibit many phenotypes: spontaneous DNA damage, chromosome loss, thermosensitivity, and acute sensitivity to genotoxic agents. These phenotypes are suppressed by mutation of histone H3 K56 into a nonacetylatable residue or by loss of K56Ac in cells lacking the histone chaperone Asf1. CONCLUSIONS Our results underscore the critical importance of Hst3/Hst4p in controlling histone H3 K56Ac and thereby maintaining chromosome integrity.
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Affiliation(s)
- Ivana Celic
- High Throughput Biology Center, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
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46
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Tanny RE, MacAlpine DM, Blitzblau HG, Bell SP. Genome-wide analysis of re-replication reveals inhibitory controls that target multiple stages of replication initiation. Mol Biol Cell 2006; 17:2415-23. [PMID: 16525018 PMCID: PMC1446079 DOI: 10.1091/mbc.e05-11-1037] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
DNA replication must be tightly controlled during each cell cycle to prevent unscheduled replication and ensure proper genome maintenance. The currently known controls that prevent re-replication act redundantly to inhibit pre-replicative complex (pre-RC) assembly outside of the G1-phase of the cell cycle. The yeast Saccharomyces cerevisiae has been a useful model organism to study how eukaryotic cells prevent replication origins from reinitiating during a single cell cycle. Using a re-replication-sensitive strain and DNA microarrays, we map sites across the S. cerevisiae genome that are re-replicated as well as sites of pre-RC formation during re-replication. Only a fraction of the genome is re-replicated by a subset of origins, some of which are capable of multiple reinitiation events. Translocation experiments demonstrate that origin-proximal sequences are sufficient to predispose an origin to re-replication. Origins that reinitiate are largely limited to those that can recruit Mcm2-7 under re-replicating conditions; however, the formation of a pre-RC is not sufficient for reinitiation. Our findings allow us to categorize origins with respect to their propensity to reinitiate and demonstrate that pre-RC formation is not the only target for the mechanisms that prevent genomic re-replication.
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Affiliation(s)
- Robyn E Tanny
- Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
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Green BM, Morreale RJ, Ozaydin B, Derisi JL, Li JJ. Genome-wide mapping of DNA synthesis in Saccharomyces cerevisiae reveals that mechanisms preventing reinitiation of DNA replication are not redundant. Mol Biol Cell 2006; 17:2401-14. [PMID: 16481397 PMCID: PMC1446083 DOI: 10.1091/mbc.e05-11-1043] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
To maintain genomic stability, reinitiation of eukaryotic DNA replication within a single cell cycle is blocked by multiple mechanisms that inactivate or remove replication proteins after G1 phase. Consistent with the prevailing notion that these mechanisms are redundant, we previously showed that simultaneous deregulation of three replication proteins, ORC, Cdc6, and Mcm2-7, was necessary to cause detectable bulk re-replication in G2/M phase in Saccharomyces cerevisiae. In this study, we used microarray comparative genomic hybridization (CGH) to provide a more comprehensive and detailed analysis of re-replication. This genome-wide analysis suggests that reinitiation in G2/M phase primarily occurs at a subset of both active and latent origins, but is independent of chromosomal determinants that specify the use and timing of these origins in S phase. We demonstrate that re-replication can be induced within S phase, but differs in amount and location from re-replication in G2/M phase, illustrating the dynamic nature of DNA replication controls. Finally, we show that very limited re-replication can be detected by microarray CGH when only two replication proteins are deregulated, suggesting that the mechanisms blocking re-replication are not redundant. Therefore we propose that eukaryotic re-replication at levels below current detection limits may be more prevalent and a greater source of genomic instability than previously appreciated.
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Affiliation(s)
- Brian M Green
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143-2200, USA
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48
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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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49
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Remus D, Blanchette M, Rio DC, Botchan MR. CDK phosphorylation inhibits the DNA-binding and ATP-hydrolysis activities of the Drosophila origin recognition complex. J Biol Chem 2005; 280:39740-51. [PMID: 16188887 DOI: 10.1074/jbc.m508515200] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2 x cyclin E and Cdk1 x cyclin B in vitro. Such phosphorylation of DmORC by Cdk2 x cyclin E, but not by Cdk1 x cyclin B, requires an "RXL" motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.
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Affiliation(s)
- Dirk Remus
- Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3204, USA
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50
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Archambault V, Ikui AE, Drapkin BJ, Cross FR. Disruption of mechanisms that prevent rereplication triggers a DNA damage response. Mol Cell Biol 2005; 25:6707-21. [PMID: 16024805 PMCID: PMC1190345 DOI: 10.1128/mcb.25.15.6707-6721.2005] [Citation(s) in RCA: 64] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Eukaryotes replicate DNA once and only once per cell cycle due to multiple, partially overlapping mechanisms efficiently preventing reinitiation. The consequences of reinitiation are unknown. Here we show that the induction of rereplication by mutations in components of the prereplicative complex (origin recognition complex [ORC], Cdc6, and minichromosome maintenance proteins) causes a cell cycle arrest with activated Rad53, a large-budded morphology, and an undivided nucleus. Combining a mutation disrupting the Clb5-Orc6 interaction (ORC6-rxl) and a mutation stabilizing Cdc6 (CDC6(Delta)NT) causes a cell cycle delay with a similar phenotype, although this background is only partially compromised for rereplication control and does not exhibit overreplication detectable by fluorescence-activated cell sorting. We conducted a systematic screen that identified genetic requirements for the viability of these cells. ORC6-rxl CDC6(Delta)NT cells depend heavily on genes required for the DNA damage response and for double-strand-break repair by homologous recombination. Our results implicate an Mre11-Mec1-dependent pathway in limiting the extent of rereplication.
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Affiliation(s)
- Vincent Archambault
- The Rockefeller University, 1230 York Ave., Box 237, New York, NY 10021, USA
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