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Azadegan C, Santoro J, Whetstine JR. CONNECTING THE DOTS: EPIGENETIC REGULATION OF EXTRACHROMOSOMAL AND INHERITED DNA AMPLIFICATIONS. J Biol Chem 2025:108454. [PMID: 40154613 DOI: 10.1016/j.jbc.2025.108454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2024] [Revised: 03/20/2025] [Accepted: 03/22/2025] [Indexed: 04/01/2025] Open
Abstract
DNA amplification has intrigued scientists for decades. Since its discovery, significant progress has been made in understanding the mechanisms promoting DNA amplification and their associated function(s). While DNA copy gains were once thought to be regulated purely by stochastic processes, recent findings have revealed the important role of epigenetic modifications in driving these amplifications and their integration into the genome. Furthermore, advances in genomic technology have enabled detailed characterization of these genomic events in terms of size, structure, formation, and regulation. This review highlights how our understanding of DNA amplifications has evolved over time, tracing its trajectory from initial discovery to the contemporary landscape. We describe how recent discoveries have started to uncover how these genomic events occur by controlled biological processes rather than stochastic mechanisms, presenting opportunities for therapeutic modulation.
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Affiliation(s)
- Chloe Azadegan
- Drexel University, College of Medicine, Philadelphia, PA, 19111; Cancer Epigenetics Institute, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA; Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA; Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia PA, 19111
| | - John Santoro
- Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA; Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia PA, 19111
| | - Johnathan R Whetstine
- Cancer Epigenetics Institute, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA; Nuclear Dynamics and Cancer Program, Fox Chase Cancer Center, Philadelphia, PA, 19111, USA; Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia PA, 19111.
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2
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Jones RM, Ruiz JH, Scaramuzza S, Nath S, Liu C, Henklewska M, Natsume T, Bristow RG, Romero F, Kanemaki MT, Gambus A. Characterizing replisome disassembly in human cells. iScience 2024; 27:110260. [PMID: 39055910 PMCID: PMC11269944 DOI: 10.1016/j.isci.2024.110260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 03/22/2024] [Accepted: 06/10/2024] [Indexed: 07/28/2024] Open
Abstract
To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.
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Affiliation(s)
- Rebecca M. Jones
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Joaquin Herrero Ruiz
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Shaun Scaramuzza
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Sarmi Nath
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Chaoyu Liu
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Marta Henklewska
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
| | - Toyoaki Natsume
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
| | - Robert G. Bristow
- Cancer Research UK – Manchester Institute, Manchester Cancer Research Center, Manchester, UK
| | - Francisco Romero
- Department of Microbiology, University of Seville, Seville, Spain
| | - Masato T. Kanemaki
- Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems, Mishima, Shizuoka, Japan
- Department of Genetics, The Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan
- Department of Biological Science, The University of Tokyo, Tokyo, Japan
| | - Agnieszka Gambus
- Institute of Cancer and Genomic Sciences, Birmingham Centre for Genome Biology, University of Birmingham, Birmingham, UK
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3
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Muñoz S, Blanco-Romero E, González-Acosta D, Rodriguez-Acebes S, Megías D, Lopes M, Méndez J. RAD51 restricts DNA over-replication from re-activated origins. EMBO J 2024; 43:1043-1064. [PMID: 38360996 PMCID: PMC10942984 DOI: 10.1038/s44318-024-00038-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2023] [Revised: 01/11/2024] [Accepted: 01/12/2024] [Indexed: 02/17/2024] Open
Abstract
Eukaryotic cells rely on several mechanisms to ensure that the genome is duplicated precisely once in each cell division cycle, preventing DNA over-replication and genomic instability. Most of these mechanisms limit the activity of origin licensing proteins to prevent the reactivation of origins that have already been used. Here, we have investigated whether additional controls restrict the extension of re-replicated DNA in the event of origin re-activation. In a genetic screening in cells forced to re-activate origins, we found that re-replication is limited by RAD51 and enhanced by FBH1, a RAD51 antagonist. In the presence of chromatin-bound RAD51, forks stemming from re-fired origins are slowed down, leading to frequent events of fork reversal. Eventual re-initiation of DNA synthesis mediated by PRIMPOL creates ssDNA gaps that facilitate the partial elimination of re-duplicated DNA by MRE11 exonuclease. In the absence of RAD51, these controls are abrogated and re-replication forks progress much longer than in normal conditions. Our study uncovers a safeguard mechanism to protect genome stability in the event of origin reactivation.
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Affiliation(s)
- Sergio Muñoz
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
| | - Elena Blanco-Romero
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
| | - Daniel González-Acosta
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Sara Rodriguez-Acebes
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
| | - Diego Megías
- Confocal Microscopy Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain
- Advanced Optical Microscopy Unit, Central Core Facilities, Instituto de Salud Carlos III, Madrid, Spain
| | - Massimo Lopes
- Institute of Molecular Cancer Research, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland
| | - Juan Méndez
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), Melchor Fernández Almagro 3, 28029, Madrid, Spain.
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4
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Mallick Ganguly O, Moulik S. Interactions of Mn complexes with DNA: the relevance of therapeutic applications towards cancer treatment. Dalton Trans 2023; 52:10639-10656. [PMID: 37475585 DOI: 10.1039/d3dt00659j] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/22/2023]
Abstract
Manganese (Mn) is one of the most significant bio-metals that helps the body to form connective tissue, bones, blood clotting factors, and sex hormones. It is necessary for fat and carbohydrate metabolism, calcium absorption, blood sugar regulation, and normal brain and nerve functions. It accelerates the synthesis of proteins, vitamin C, and vitamin B. It is also involved in the catalysis of hematopoiesis, regulation of the endocrine level, and improvement of immune function. Again, Mn metalloenzymes like arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and Mn superoxide dismutase (MnSOD) contribute to the metabolism processes and reduce oxidative stress against free radicals. Recent investigations have revealed that synthetic Mn-complexes act as antibacterial and antifungal agents. As a result, chemists and biologists have been actively involved in developing Mn-based drugs for the treatment of various diseases including cancer. Therefore, any therapeutic drugs based on manganese complexes would be invaluable for the treatment of cancer/infectious diseases and could be a better substitute for cisplatin and other related platinum based chemotherapeutic drugs. From this perspective, attempts have been made to discuss the interactions and nuclease activities of Mn(II/III/IV) complexes with DNA through which one can evaluate their therapeutic applications.
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Affiliation(s)
- Oishi Mallick Ganguly
- St Xavier's College, 30, Park St, Mullick Bazar, Park Street area, Kolkata, West Bengal 700016, India
| | - Shuvojit Moulik
- Suraksha Diagnostics Pvt Ltd, Newtown 12/1, Premises No. 02-0327, DG Block(Newtown), Action Area 1D, Newtown, Kolkata, West Bengal 700156, India.
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5
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Ratnayeke N, Baris Y, Chung M, Yeeles JTP, Meyer T. CDT1 inhibits CMG helicase in early S phase to separate origin licensing from DNA synthesis. Mol Cell 2023; 83:26-42.e13. [PMID: 36608667 DOI: 10.1016/j.molcel.2022.12.004] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2022] [Revised: 09/16/2022] [Accepted: 12/08/2022] [Indexed: 01/07/2023]
Abstract
Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4Cdt2 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.
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Affiliation(s)
- Nalin Ratnayeke
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA
| | - Yasemin Baris
- Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK
| | - Mingyu Chung
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Joseph T P Yeeles
- Laboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, UK
| | - Tobias Meyer
- Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10065, USA.
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6
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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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7
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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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8
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Saxena S, Zou L. Hallmarks of DNA replication stress. Mol Cell 2022; 82:2298-2314. [PMID: 35714587 DOI: 10.1016/j.molcel.2022.05.004] [Citation(s) in RCA: 186] [Impact Index Per Article: 62.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Revised: 04/15/2022] [Accepted: 05/04/2022] [Indexed: 12/12/2022]
Abstract
Faithful DNA replication is critical for the maintenance of genomic integrity. Although DNA replication machinery is highly accurate, the process of DNA replication is constantly challenged by DNA damage and other intrinsic and extrinsic stresses throughout the genome. A variety of cellular stresses interfering with DNA replication, which are collectively termed replication stress, pose a threat to genomic stability in both normal and cancer cells. To cope with replication stress and maintain genomic stability, cells have evolved a complex network of cellular responses to alleviate and tolerate replication problems. This review will focus on the major sources of replication stress, the impacts of replication stress in cells, and the assays to detect replication stress, offering an overview of the hallmarks of DNA replication stress.
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Affiliation(s)
- Sneha Saxena
- Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA
| | - Lee Zou
- Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
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9
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Zaffar E, Ferreira P, Sanchez-Pulido L, Boos D. The Role of MTBP as a Replication Origin Firing Factor. BIOLOGY 2022; 11:biology11060827. [PMID: 35741348 PMCID: PMC9219753 DOI: 10.3390/biology11060827] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/29/2022] [Revised: 05/20/2022] [Accepted: 05/22/2022] [Indexed: 12/12/2022]
Abstract
The initiation step of replication at replication origins determines when and where in the genome replication machines, replisomes, are generated. Tight control of replication initiation helps facilitate the two main tasks of genome replication, to duplicate the genome accurately and exactly once each cell division cycle. The regulation of replication initiation must ensure that initiation occurs during the S phase specifically, that no origin fires more than once per cell cycle, that enough origins fire to avoid non-replicated gaps, and that the right origins fire at the right time but only in favorable circumstances. Despite its importance for genetic homeostasis only the main molecular processes of eukaryotic replication initiation and its cellular regulation are understood. The MTBP protein (Mdm2-binding protein) is so far the last core replication initiation factor identified in metazoan cells. MTBP is the orthologue of yeast Sld7. It is essential for origin firing, the maturation of pre-replicative complexes (pre-RCs) into replisomes, and is emerging as a regulation focus targeted by kinases and by regulated degradation. We present recent insight into the structure and cellular function of the MTBP protein in light of recent structural and biochemical studies revealing critical molecular details of the eukaryotic origin firing reaction. How the roles of MTBP in replication and other cellular processes are mutually connected and are related to MTBP's contribution to tumorigenesis remains largely unclear.
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Affiliation(s)
- Eman Zaffar
- Molecular Genetics II, Centre for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany; (E.Z.); (P.F.)
| | - Pedro Ferreira
- Molecular Genetics II, Centre for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany; (E.Z.); (P.F.)
| | - Luis Sanchez-Pulido
- Medical Research Council Human Genetics Unit, IGC, University of Edinburgh, Edinburgh EH9 3JR, UK;
| | - Dominik Boos
- Molecular Genetics II, Centre for Medical Biotechnology, University of Duisburg-Essen, 45141 Essen, Germany; (E.Z.); (P.F.)
- Correspondence: ; Tel.: +49-201-183-4132
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10
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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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11
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Abstract
Cancer is a group of diseases in which cells divide continuously and excessively. Cell division is tightly regulated by multiple evolutionarily conserved cell cycle control mechanisms, to ensure the production of two genetically identical cells. Cell cycle checkpoints operate as DNA surveillance mechanisms that prevent the accumulation and propagation of genetic errors during cell division. Checkpoints can delay cell cycle progression or, in response to irreparable DNA damage, induce cell cycle exit or cell death. Cancer-associated mutations that perturb cell cycle control allow continuous cell division chiefly by compromising the ability of cells to exit the cell cycle. Continuous rounds of division, however, create increased reliance on other cell cycle control mechanisms to prevent catastrophic levels of damage and maintain cell viability. New detailed insights into cell cycle control mechanisms and their role in cancer reveal how these dependencies can be best exploited in cancer treatment.
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Affiliation(s)
- Helen K Matthews
- MRC Laboratory for Molecular Cell Biology, University College London, London, UK
- Department of Biomedical Science, University of Sheffield, Sheffield, UK
| | - Cosetta Bertoli
- MRC Laboratory for Molecular Cell Biology, University College London, London, UK
| | - Robertus A M de Bruin
- MRC Laboratory for Molecular Cell Biology, University College London, London, UK.
- UCL Cancer Institute, University College London, London, UK.
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12
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Arenas V, Castaño JL, Domínguez-García JJ, Yáñez L, Pipaón C. A Different View for an Old Disease: NEDDylation and Other Ubiquitin-Like Post-Translational Modifications in Chronic Lymphocytic Leukemia. Front Oncol 2021; 11:729550. [PMID: 34631557 PMCID: PMC8495217 DOI: 10.3389/fonc.2021.729550] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Accepted: 09/07/2021] [Indexed: 12/03/2022] Open
Abstract
Despite the enormous amount of molecular data obtained over the years, the molecular etiology of chronic lymphocytic leukemia (CLL) is still largely unknown. All that information has enabled the development of new therapeutic approaches that have improved life expectancy of the patients but are still not curative. We must increase our knowledge of the molecular alterations responsible for the characteristics common to all CLL patients. One of such characteristics is the poor correlation between mRNA and protein expression, that suggests a role of post-translational mechanisms in CLL physiopathology. Drugs targeting these processes have indeed demonstrated an effect either alone or in combination with other aimed at specific pathways. A recent article unveiled an increment in ubiquitin-like modifications in CLL, with many protein members of relevant pathways affected. Interestingly, the inhibition of the NEDD8-activating protein NAE reverted a substantial number of those modifications. The present review gets the scarce data published about the role of NEDDylation in CLL together and establishes connections to what is known from other neoplasias, thus providing a new perspective to the underlying mechanisms in CLL.
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Affiliation(s)
- Víctor Arenas
- Laboratorio de Hematología Molecular, Servicio de Hematología, Hospital Universitario Marqués de Valdecilla-Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Jose Luis Castaño
- Laboratorio de Hematología Molecular, Servicio de Hematología, Hospital Universitario Marqués de Valdecilla-Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Juan José Domínguez-García
- Laboratorio de Hematología Molecular, Servicio de Hematología, Hospital Universitario Marqués de Valdecilla-Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Lucrecia Yáñez
- Laboratorio de Hematología Molecular, Servicio de Hematología, Hospital Universitario Marqués de Valdecilla-Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
| | - Carlos Pipaón
- Laboratorio de Hematología Molecular, Servicio de Hematología, Hospital Universitario Marqués de Valdecilla-Instituto de Investigación Marqués de Valdecilla (IDIVAL), Santander, Spain
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13
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SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition. Nat Commun 2021; 12:5779. [PMID: 34599168 PMCID: PMC8486843 DOI: 10.1038/s41467-021-26049-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 09/09/2021] [Indexed: 12/28/2022] Open
Abstract
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy. Geminin-Cdt1 plays essential roles in the regulation of DNA replication. Here the authors reveal that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP prevents DNA replication over-firing and genome instability by affecting Geminin ubiquitination.
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14
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Abstract
Safeguards against excess DNA replication are often dysregulated in cancer, and driving cancer cells towards over-replication is a promising therapeutic strategy. We determined DNA synthesis patterns in cancer cells undergoing partial genome re-replication due to perturbed regulatory interactions (re-replicating cells). These cells exhibited slow replication, increased frequency of replication initiation events, and a skewed initiation pattern that preferentially reactivated early-replicating origins. Unlike in cells exposed to replication stress, which activated a novel group of hitherto unutilized (dormant) replication origins, the preferred re-replicating origins arose from the same pool of potential origins as those activated during normal growth. Mechanistically, the skewed initiation pattern reflected a disproportionate distribution of pre-replication complexes on distinct regions of licensed chromatin prior to replication. This distinct pattern suggests that circumventing the strong inhibitory interactions that normally prevent excess DNA synthesis can occur via at least two pathways, each activating a distinct set of replication origins.
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15
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Tian J, Lu Z, Niu S, Zhang S, Ying P, Wang L, Zhang M, Cai Y, Dong T, Zhu Y, Zhong R, Wang Z, Chang J, Miao X. Aberrant MCM10 SUMOylation induces genomic instability mediated by a genetic variant associated with survival of esophageal squamous cell carcinoma. Clin Transl Med 2021; 11:e485. [PMID: 34185429 PMCID: PMC8236122 DOI: 10.1002/ctm2.485] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2021] [Revised: 06/13/2021] [Accepted: 06/16/2021] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Esophageal squamous cell carcinoma (ESCC) is one of the common gastrointestinal malignancy with an inferior prognosis outcome. DNA replication licensing aberration induced by dysregulation of minichromosome maintenance proteins (MCMs) causes genomic instability and cancer metastasis. SUMOylation modification plays a pivotal role in regulation of genomic integrity, while its dysregulation fueled by preexisting germline variants in cancers remains poorly understood. METHODS Firstly, we conducted two-stage survival analysis consisting of an exome-wide association study in 904 ESCC samples and another independent 503 ESCC samples. Then, multipronged functional experiments were performed to illuminate the potential biological mechanisms underlying the promising variants, and MCM10 influences the ESCC progression. Finally, we tested the effects of MCM10 inhibitors on ESCC cells. RESULTS A germline variant rs2274110 located at the exon 15 of MCM10 was identified to be significantly associated with the prognosis of ESCC patients. Individuals carrying rs2274110-AA genotypes confer a poor survival (hazard ratio = 1.61, 95% confidence interval = 1.35-1.93, p = 1.35 × 10-7 ), compared with subjects carrying rs2274110-AG/GG genotypes. Furthermore, we interestingly found that the variant can increase SUMOylation levels at K669 site (Lys[K]699Arg[R]) of MCM10 protein mediated by SUMO2/3 enzymes, which resulted in an aberrant overexpression of MCM10. Mechanistically, aberrant overexpression of MCM10 facilitated the proliferation and metastasis abilities of ESCC cells in vitro and in vivo by inducing DNA over-replication and genomic instability, providing functional evidence to support our population finding that high expression of MCM10 is extensively presented in tumor tissues of ESCC and correlated with inferior survival outcomes of multiple cancer types, including ESCC. Finally, MCM10 inhibitors Suramin and its analogues were revealed to effectively block the metastasis of ESCC cells. CONCLUSIONS These findings not only demonstrate a potential biological mechanism between aberrant SUMOylation, genomic instability and cancer metastasis, but also provide a promising biomarker aiding in stratifying ESCC individuals with different prognosis, as well as a potential therapeutic target MCM10.
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Affiliation(s)
- Jianbo Tian
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Zequn Lu
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Siyuan Niu
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Shanshan Zhang
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Pingting Ying
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Lu Wang
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Ming Zhang
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Yimin Cai
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Tianyi Dong
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Ying Zhu
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Rong Zhong
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Zhihua Wang
- Department of UrologyTongji HospitalTongji Medical CollegeHuazhong University of Science and TechnologyWuhanChina
| | - Jiang Chang
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
| | - Xiaoping Miao
- Department of Epidemiology and BiostatisticsKey Laboratory for Environment and HealthSchool of Public HealthTongji Medical CollegeHuazhong University of Sciences and TechnologyWuhanChina
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16
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Hammond-Martel I, Verreault A, Wurtele H. Chromatin dynamics and DNA replication roadblocks. DNA Repair (Amst) 2021; 104:103140. [PMID: 34087728 DOI: 10.1016/j.dnarep.2021.103140] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 05/18/2021] [Accepted: 05/20/2021] [Indexed: 11/27/2022]
Abstract
A broad spectrum of spontaneous and genotoxin-induced DNA lesions impede replication fork progression. The DNA damage response that acts to promote completion of DNA replication is associated with dynamic changes in chromatin structure that include two distinct processes which operate genome-wide during S-phase. The first, often referred to as histone recycling or parental histone segregation, is characterized by the transfer of parental histones located ahead of replication forks onto nascent DNA. The second, known as de novo chromatin assembly, consists of the deposition of new histone molecules onto nascent DNA. Because these two processes occur at all replication forks, their potential to influence a multitude of DNA repair and DNA damage tolerance mechanisms is considerable. The purpose of this review is to provide a description of parental histone segregation and de novo chromatin assembly, and to illustrate how these processes influence cellular responses to DNA replication roadblocks.
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Affiliation(s)
- Ian Hammond-Martel
- Centre de recherche de l'Hôpital Maisonneuve-Rosemont, 5415 boulevard de l'Assomption, Montreal, H1T 2M4, Canada
| | - Alain Verreault
- Institute for Research in Immunology and Cancer, Université de Montréal, P.O. Box 6128, Succursale Centre-Ville, Montreal, H3C 3J7, Canada; Département de Pathologie et Biologie Cellulaire, Université de Montréal, 2900 Edouard Montpetit Blvd, Montreal, H3T 1J4, Canada
| | - Hugo Wurtele
- Centre de recherche de l'Hôpital Maisonneuve-Rosemont, 5415 boulevard de l'Assomption, Montreal, H1T 2M4, Canada; Département de Médecine, Université de Montréal, Université de Montréal, 2900 Edouard Montpetit Blvd, Montreal, H3T 1J4, Canada.
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17
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Rapsomaniki MA, Maxouri S, Nathanailidou P, Garrastacho MR, Giakoumakis NN, Taraviras S, Lygeros J, Lygerou Z. In silico analysis of DNA re-replication across a complete genome reveals cell-to-cell heterogeneity and genome plasticity. NAR Genom Bioinform 2021; 3:lqaa112. [PMID: 33554116 PMCID: PMC7846089 DOI: 10.1093/nargab/lqaa112] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 12/15/2020] [Accepted: 01/20/2021] [Indexed: 01/06/2023] Open
Abstract
DNA replication is a complex and remarkably robust process: despite its inherent uncertainty, manifested through stochastic replication timing at a single-cell level, multiple control mechanisms ensure its accurate and timely completion across a population. Disruptions in these mechanisms lead to DNA re-replication, closely connected to genomic instability and oncogenesis. Here, we present a stochastic hybrid model of DNA re-replication that accurately portrays the interplay between discrete dynamics, continuous dynamics and uncertainty. Using experimental data on the fission yeast genome, model simulations show how different regions respond to re-replication and permit insight into the key mechanisms affecting re-replication dynamics. Simulated and experimental population-level profiles exhibit a good correlation along the genome, robust to model parameters, validating our approach. At a single-cell level, copy numbers of individual loci are affected by intrinsic properties of each locus, in cis effects from adjoining loci and in trans effects from distant loci. In silico analysis and single-cell imaging reveal that cell-to-cell heterogeneity is inherent in re-replication and can lead to genome plasticity and a plethora of genotypic variations.
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Affiliation(s)
- Maria Anna Rapsomaniki
- Department of Biology, School of Medicine, University of Patras, 26500 Rio Patras, Greece
| | - Stella Maxouri
- Department of Biology, School of Medicine, University of Patras, 26500 Rio Patras, Greece
| | - Patroula Nathanailidou
- Department of Biology, School of Medicine, University of Patras, 26500 Rio Patras, Greece
| | | | | | - Stavros Taraviras
- Department of Physiology, School of Medicine, University of Patras, 26500 Rio Patras, Greece
| | - John Lygeros
- Automatic Control Laboratory, ETH Zurich, 8092 Zurich, Switzerland
| | - Zoi Lygerou
- Department of Biology, School of Medicine, University of Patras, 26500 Rio Patras, Greece
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18
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Under-Replicated DNA: The Byproduct of Large Genomes? Cancers (Basel) 2020; 12:cancers12102764. [PMID: 32992928 PMCID: PMC7601121 DOI: 10.3390/cancers12102764] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2020] [Revised: 09/21/2020] [Accepted: 09/22/2020] [Indexed: 12/28/2022] Open
Abstract
In this review, we provide an overview of how proliferating eukaryotic cells overcome one of the main threats to genome stability: incomplete genomic DNA replication during S phase. We discuss why it is currently accepted that double fork stalling (DFS) events are unavoidable events in higher eukaryotes with large genomes and which responses have evolved to cope with its main consequence: the presence of under-replicated DNA (UR-DNA) outside S phase. Particular emphasis is placed on the processes that constrain the detrimental effects of UR-DNA. We discuss how mitotic DNA synthesis (MiDAS), mitotic end joining events and 53BP1 nuclear bodies (53BP1-NBs) deal with such specific S phase DNA replication remnants during the subsequent phases of the cell cycle.
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19
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Origin of Genome Instability and Determinants of Mutational Landscape in Cancer Cells. Genes (Basel) 2020; 11:genes11091101. [PMID: 32967144 PMCID: PMC7563369 DOI: 10.3390/genes11091101] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2020] [Revised: 09/12/2020] [Accepted: 09/18/2020] [Indexed: 12/31/2022] Open
Abstract
Genome instability is a crucial and early event associated with an increased predisposition to tumor formation. In the absence of any exogenous agent, a single human cell is subjected to about 70,000 DNA lesions each day. It has now been shown that physiological cellular processes including DNA transactions during DNA replication and transcription contribute to DNA damage and induce DNA damage responses in the cell. These processes are also influenced by the three dimensional-chromatin architecture and epigenetic regulation which are altered during the malignant transformation of cells. In this review, we have discussed recent insights about how replication stress, oncogene activation, chromatin dynamics, and the illegitimate recombination of cell-free chromatin particles deregulate cellular processes in cancer cells and contribute to their evolution. The characterization of such endogenous sources of genome instability in cancer cells can be exploited for the development of new biomarkers and more effective therapies for cancer treatment.
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20
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Menzel J, Tatman P, Black JC. Isolation and analysis of rereplicated DNA by Rerep-Seq. Nucleic Acids Res 2020; 48:e58. [PMID: 32239215 PMCID: PMC7261181 DOI: 10.1093/nar/gkaa197] [Citation(s) in RCA: 59] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Revised: 03/05/2020] [Accepted: 03/16/2020] [Indexed: 01/31/2023] Open
Abstract
Changes in gene copy number contribute to genomic instability, the onset and progression of cancer, developmental abnormalities and adaptive potential. The origins of gene amplifications have remained elusive; however, DNA rereplication has been implicated as a source of gene amplifications. The inability to determine which sequences are rereplicated and under what conditions have made it difficult to determine the validity of the proposed models. Here we present Rerep-Seq, a technique that selectively enriches for rereplicated DNA in preparation for analysis by DNA sequencing that can be applied to any species. We validated Rerep-Seq by simulating DNA rereplication in yeast and human cells. Using Rerep-Seq, we demonstrate that rereplication induced in Saccharomyces cerevisiae by deregulated origin licensing is non-random and defined by broad domains that span multiple replication origins and topological boundaries.
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Affiliation(s)
- Johannes Menzel
- University of Colorado Anschutz Medical Campus, Department of Pharmacology, 12800 E 19th Ave, Aurora, CO 80045, USA.,University of Colorado Anschutz Medical Campus, Molecular Biology Graduate Program, 12800 E 19th Ave, Aurora, CO 80045, USA
| | - Philip Tatman
- University of Colorado Anschutz Medical Campus, Department of Pharmacology, 12800 E 19th Ave, Aurora, CO 80045, USA.,University of Colorado Anschutz Medical Campus, Medical Scientist Training Program, 12800 E 19th Ave, Aurora, CO 80045, USA
| | - Joshua C Black
- University of Colorado Anschutz Medical Campus, Department of Pharmacology, 12800 E 19th Ave, Aurora, CO 80045, USA.,University of Colorado Anschutz Medical Campus, Molecular Biology Graduate Program, 12800 E 19th Ave, Aurora, CO 80045, USA.,University of Colorado Anschutz Medical Campus, Medical Scientist Training Program, 12800 E 19th Ave, Aurora, CO 80045, USA
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21
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Targeting ubiquitin-activating enzyme induces ER stress-mediated apoptosis in B-cell lymphoma cells. Blood Adv 2020; 3:51-62. [PMID: 30617217 DOI: 10.1182/bloodadvances.2018026880] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2018] [Accepted: 11/29/2018] [Indexed: 12/26/2022] Open
Abstract
Alterations in the ubiquitin proteasome system (UPS) leave malignant cells in heightened cellular stress, making them susceptible to proteasome inhibition. However, given the limited efficacy of proteasome inhibitors in non-Hodgkin lymphoma (NHL), novel approaches to target the UPS are needed. Here, we show that TAK-243, the first small-molecule inhibitor of the ubiquitin activating enzyme (UAE) to enter clinical development, disrupts all ubiquitin signaling and global protein ubiquitination in diffuse large B-cell lymphoma (DLBCL) cells, thereby inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Activation of the ER stress response protein kinase R (PKR)-like ER kinase and phosphorylation of eukaryotic translation initiator factor 2α led to upregulation of the proapoptotic molecule C/EBP homologous protein and cell death across a panel of DLBCL cell lines independent of cell of origin. Concurrently, targeting UAE led to accumulation of Cdt1, a replication licensing factor, leading to DNA rereplication, checkpoint activation, and cell cycle arrest. MYC oncoprotein sensitized DLBCL cells to UAE inhibition; engineered expression of MYC enhanced while genetic MYC knockdown protected from TAK-243-induced apoptosis. UAE inhibition demonstrated enhanced ER stress and UPR and increased potency compared with bortezomib in DLBCL cell lines. In vivo treatment with TAK-243 restricted the growth of xenografted DLBCL tumors, accompanied by reduced cell proliferation and apoptosis. Finally, primary patient-derived DLBCL cells, including those expressing aberrant MYC, demonstrated susceptibility to UAE inhibition. In sum, targeting UAE may hold promise as a novel therapeutic approach in NHL.
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22
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Grant GD, Kedziora KM, Limas JC, Cook JG, Purvis JE. Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI. Cell Cycle 2019; 17:2496-2516. [PMID: 30421640 DOI: 10.1080/15384101.2018.1547001] [Citation(s) in RCA: 72] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions.
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Affiliation(s)
- Gavin D Grant
- a Department of Biochemistry and Biophysics , The University of North Carolina , Chapel Hill , NC , USA.,b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA
| | - Katarzyna M Kedziora
- c Department of Genetics , The University of North Carolina , Chapel Hill , NC , USA
| | - Juanita C Limas
- d Department of Pharmacology , The University of North Carolina , Chapel Hill , NC , USA
| | - Jeanette Gowen Cook
- a Department of Biochemistry and Biophysics , The University of North Carolina , Chapel Hill , NC , USA.,b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA.,d Department of Pharmacology , The University of North Carolina , Chapel Hill , NC , USA
| | - Jeremy E Purvis
- b Lineberger Comprehensive Cancer Center , The University of North Carolina , Chapel Hill , NC , USA.,c Department of Genetics , The University of North Carolina , Chapel Hill , NC , USA
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23
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Petropoulos M, Champeris Tsaniras S, Taraviras S, Lygerou Z. Replication Licensing Aberrations, Replication Stress, and Genomic Instability. Trends Biochem Sci 2019; 44:752-764. [PMID: 31054805 DOI: 10.1016/j.tibs.2019.03.011] [Citation(s) in RCA: 77] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2019] [Revised: 03/24/2019] [Accepted: 03/27/2019] [Indexed: 01/07/2023]
Abstract
Strict regulation of DNA replication is of fundamental significance for the maintenance of genome stability. Licensing of origins of DNA replication is a critical event for timely genome duplication. Errors in replication licensing control lead to genomic instability across evolution. Here, we present accumulating evidence that aberrant replication licensing is linked to oncogene-induced replication stress and poses a major threat to genome stability, promoting tumorigenesis. Oncogene activation can lead to defects in where along the genome and when during the cell cycle licensing takes place, resulting in replication stress. We also discuss the potential of replication licensing as a specific target for novel anticancer therapies.
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Affiliation(s)
- Michalis Petropoulos
- Department of Biology, School of Medicine, University of Patras, Patras 26504, Greece
| | | | - Stavros Taraviras
- Department of Physiology, School of Medicine, University of Patras, Patras 26504, Greece.
| | - Zoi Lygerou
- Department of Biology, School of Medicine, University of Patras, Patras 26504, Greece.
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24
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Mughal MJ, Mahadevappa R, Kwok HF. DNA replication licensing proteins: Saints and sinners in cancer. Semin Cancer Biol 2018; 58:11-21. [PMID: 30502375 DOI: 10.1016/j.semcancer.2018.11.009] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2018] [Revised: 11/08/2018] [Accepted: 11/26/2018] [Indexed: 12/12/2022]
Abstract
DNA replication is all-or-none process in the cell, meaning, once the DNA replication begins it proceeds to completion. Hence, to achieve maximum control of DNA replication, eukaryotic cells employ a multi-subunit initiator protein complex known as "pre-replication complex or DNA replication licensing complex (DNA replication LC). This complex involves multiple proteins which are origin-recognition complex family proteins, cell division cycle-6, chromatin licensing and DNA replication factor 1, and minichromosome maintenance family proteins. Higher-expression of DNA replication LC proteins appears to be an early event during development of cancer since it has been a common hallmark observed in a wide variety of cancers such as oesophageal, laryngeal, pulmonary, mammary, colorectal, renal, urothelial etc. However, the exact mechanisms leading to the abnormally high expression of DNA replication LC have not been clearly deciphered. Increased expression of DNA replication LC leads to licensing and/or firing of multiple origins thereby inducing replication stress and genomic instability. Therapeutic approaches where the reduction in the activity of DNA replication LC was achieved either by siRNA or shRNA techniques, have shown increased sensitivity of cancer cell lines towards the anti-cancer drugs such as cisplatin, 5-Fluorouracil, hydroxyurea etc. Thus, the expression level of DNA replication LC within the cell determines a cell's fate thereby creating a paradox where DNA replication LC acts as both "Saint" and "Sinner". With a potential to increase sensitivity to chemotherapy drugs, DNA replication LC proteins have prospective clinical importance in fighting cancer. Hence, in this review, we will shed light on importance of DNA replication LC with an aim to use DNA replication LC in diagnosis and prognosis of cancer in patients as well as possible therapeutic targets for cancer therapy.
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Affiliation(s)
- Muhammad Jameel Mughal
- Cancer Centre, Faculty of Health Sciences, University of Macau, Avenida de Universidade, Taipa, Macau
| | - Ravikiran Mahadevappa
- Cancer Centre, Faculty of Health Sciences, University of Macau, Avenida de Universidade, Taipa, Macau
| | - Hang Fai Kwok
- Cancer Centre, Faculty of Health Sciences, University of Macau, Avenida de Universidade, Taipa, Macau.
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25
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Tsao WC, Eckert KA. Detours to Replication: Functions of Specialized DNA Polymerases during Oncogene-induced Replication Stress. Int J Mol Sci 2018; 19:ijms19103255. [PMID: 30347795 PMCID: PMC6214091 DOI: 10.3390/ijms19103255] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2018] [Revised: 10/15/2018] [Accepted: 10/15/2018] [Indexed: 01/10/2023] Open
Abstract
Incomplete and low-fidelity genome duplication contribute to genomic instability and cancer development. Difficult-to-Replicate Sequences, or DiToRS, are natural impediments in the genome that require specialized DNA polymerases and repair pathways to complete and maintain faithful DNA synthesis. DiToRS include non B-DNA secondary structures formed by repetitive sequences, for example within chromosomal fragile sites and telomeres, which inhibit DNA replication under endogenous stress conditions. Oncogene activation alters DNA replication dynamics and creates oncogenic replication stress, resulting in persistent activation of the DNA damage and replication stress responses, cell cycle arrest, and cell death. The response to oncogenic replication stress is highly complex and must be tightly regulated to prevent mutations and tumorigenesis. In this review, we summarize types of known DiToRS and the experimental evidence supporting replication inhibition, with a focus on the specialized DNA polymerases utilized to cope with these obstacles. In addition, we discuss different causes of oncogenic replication stress and its impact on DiToRS stability. We highlight recent findings regarding the regulation of DNA polymerases during oncogenic replication stress and the implications for cancer development.
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Affiliation(s)
- Wei-Chung Tsao
- Department of Pathology, The Jake Gittlen Laboratories for Cancer Research, Hershey, PA 17033, USA.
| | - Kristin A Eckert
- Department of Pathology, The Jake Gittlen Laboratories for Cancer Research, Hershey, PA 17033, USA.
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26
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Pozo PN, Matson JP, Cole Y, Kedziora KM, Grant GD, Temple B, Cook JG. Cdt1 variants reveal unanticipated aspects of interactions with cyclin/CDK and MCM important for normal genome replication. Mol Biol Cell 2018; 29:2989-3002. [PMID: 30281379 PMCID: PMC6333176 DOI: 10.1091/mbc.e18-04-0242] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
The earliest step in DNA replication is origin licensing, which is the DNA loading of minichromosome maintenance (MCM) helicase complexes. The Cdc10-dependent transcript 1 (Cdt1) protein is essential for MCM loading during the G1 phase of the cell cycle, but the mechanism of Cdt1 function is still incompletely understood. We examined a collection of rare Cdt1 variants that cause a form of primordial dwarfism (the Meier-Gorlin syndrome) plus one hypomorphic Drosophila allele to shed light on Cdt1 function. Three hypomorphic variants load MCM less efficiently than wild-type (WT) Cdt1, and their lower activity correlates with impaired MCM binding. A structural homology model of the human Cdt1-MCM complex positions the altered Cdt1 residues at two distinct interfaces rather than the previously described single MCM interaction domain. Surprisingly, one dwarfism allele (Cdt1-A66T) is more active than WT Cdt1. This hypermorphic variant binds both cyclin A and SCFSkp2 poorly relative to WT Cdt1. Detailed quantitative live-cell imaging analysis demonstrated no change in the stability of this variant, however. Instead, we propose that cyclin A/CDK inhibits the Cdt1 licensing function independent of the creation of the SCFSkp2 phosphodegron. Together, these findings identify key Cdt1 interactions required for both efficient origin licensing and tight Cdt1 regulation to ensure normal cell proliferation and genome stability.
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Affiliation(s)
- Pedro N Pozo
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Jacob P Matson
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Yasemin Cole
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Katarzyna M Kedziora
- Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Gavin D Grant
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Brenda Temple
- Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,R. L. Juliano Structural Bioinformatics Core Facility, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Center for Structural Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
| | - Jeanette Gowen Cook
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
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27
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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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28
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Kotsantis P, Petermann E, Boulton SJ. Mechanisms of Oncogene-Induced Replication Stress: Jigsaw Falling into Place. Cancer Discov 2018; 8:537-555. [PMID: 29653955 DOI: 10.1158/2159-8290.cd-17-1461] [Citation(s) in RCA: 270] [Impact Index Per Article: 38.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2018] [Revised: 02/26/2018] [Accepted: 03/09/2018] [Indexed: 12/31/2022]
Abstract
Oncogene activation disturbs cellular processes and accommodates a complex landscape of changes in the genome that contribute to genomic instability, which accelerates mutation rates and promotes tumorigenesis. Part of this cellular turmoil involves deregulation of physiologic DNA replication, widely described as replication stress. Oncogene-induced replication stress is an early driver of genomic instability and is attributed to a plethora of factors, most notably aberrant origin firing, replication-transcription collisions, reactive oxygen species, and defective nucleotide metabolism.Significance: Replication stress is a fundamental step and an early driver of tumorigenesis and has been associated with many activated oncogenes. Deciphering the mechanisms that contribute to the replication stress response may provide new avenues for targeted cancer treatment. In this review, we discuss the latest findings on the DNA replication stress response and examine the various mechanisms through which activated oncogenes induce replication stress. Cancer Discov; 8(5); 537-55. ©2018 AACR.
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Affiliation(s)
| | - Eva Petermann
- Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
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29
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Muñoz S, Búa S, Rodríguez-Acebes S, Megías D, Ortega S, de Martino A, Méndez J. In Vivo DNA Re-replication Elicits Lethal Tissue Dysplasias. Cell Rep 2018; 19:928-938. [PMID: 28467906 DOI: 10.1016/j.celrep.2017.04.032] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Revised: 03/10/2017] [Accepted: 04/12/2017] [Indexed: 10/19/2022] Open
Abstract
Mammalian DNA replication origins are "licensed" by the loading of DNA helicases, a reaction that is mediated by CDC6 and CDT1 proteins. After initiation of DNA synthesis, CDC6 and CDT1 are inhibited to prevent origin reactivation and DNA overreplication before cell division. CDC6 and CDT1 are highly expressed in many types of cancer cells, but the impact of their deregulated expression had not been investigated in vivo. Here, we have generated mice strains that allow the conditional overexpression of both proteins. Adult mice were unharmed by the individual overexpression of either CDC6 or CDT1, but their combined deregulation led to DNA re-replication in progenitor cells and lethal tissue dysplasias. This study offers mechanistic insights into the necessary cooperation between CDC6 and CDT1 for facilitation of origin reactivation and describes the physiological consequences of DNA overreplication.
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Affiliation(s)
- Sergio Muñoz
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Sabela Búa
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Sara Rodríguez-Acebes
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Diego Megías
- Confocal Microscopy Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Sagrario Ortega
- Transgenic Mice Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Alba de Martino
- Compared Pathology Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain
| | - Juan Méndez
- DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO), 3 Melchor Fernández Almagro, 28029 Madrid, Spain.
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30
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Sankar S, Patterson E, Lewis EM, Waller LE, Tong C, Dearborn J, Wozniak D, Rubin JB, Kroll KL. Geminin deficiency enhances survival in a murine medulloblastoma model by inducing apoptosis of preneoplastic granule neuron precursors. Genes Cancer 2017; 8:725-744. [PMID: 29234490 PMCID: PMC5724806 DOI: 10.18632/genesandcancer.157] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Medulloblastoma is the most common malignant brain cancer of childhood. Further understanding of tumorigenic mechanisms may define new therapeutic targets. Geminin maintains genome fidelity by controlling re-initiation of DNA replication within a cell cycle. In some contexts, Geminin inhibition induces cancer-selective cell cycle arrest and apoptosis and/or sensitizes cancer cells to Topoisomerase IIα inhibitors such as etoposide, which is used in combination chemotherapies for medulloblastoma. However, Geminin's potential role in medulloblastoma tumorigenesis remained undefined. Here, we found that Geminin is highly expressed in human and mouse medulloblastomas and in murine granule neuron precursor (GNP) cells during cerebellar development. Conditional Geminin loss significantly enhanced survival in the SmoA1 mouse medulloblastoma model. Geminin loss in this model also reduced numbers of preneoplastic GNPs persisting at one postnatal month, while at two postnatal weeks these cells exhibited an elevated DNA damage response and apoptosis. Geminin knockdown likewise impaired human medulloblastoma cell growth, activating G2 checkpoint and DNA damage response pathways, triggering spontaneous apoptosis, and enhancing G2 accumulation of cells in response to etoposide treatment. Together, these data suggest preneoplastic and cancer cell-selective roles for Geminin in medulloblastoma, and suggest that targeting Geminin may impair tumor growth and enhance responsiveness to Topoisomerase IIα-directed chemotherapies.
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Affiliation(s)
- Savita Sankar
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
| | - Ethan Patterson
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
| | - Emily M Lewis
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
| | - Laura E Waller
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
| | - Caili Tong
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
| | - Joshua Dearborn
- Department of Psychiatry, Washington University School of Medicine, Saint Louis, MO, USA
| | - David Wozniak
- Department of Psychiatry, Washington University School of Medicine, Saint Louis, MO, USA
| | - Joshua B Rubin
- Department of Pediatrics, Washington University School of Medicine, Saint Louis, MO, USA
| | - Kristen L Kroll
- Department of Developmental Biology, Washington University School of Medicine, Saint Louis, MO, USA
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31
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Wlodek A, Kendrew SG, Coates NJ, Hold A, Pogwizd J, Rudder S, Sheehan LS, Higginbotham SJ, Stanley-Smith AE, Warneck T, Nur-E-Alam M, Radzom M, Martin CJ, Overvoorde L, Samborskyy M, Alt S, Heine D, Carter GT, Graziani EI, Koehn FE, McDonald L, Alanine A, Rodríguez Sarmiento RM, Chao SK, Ratni H, Steward L, Norville IH, Sarkar-Tyson M, Moss SJ, Leadlay PF, Wilkinson B, Gregory MA. Diversity oriented biosynthesis via accelerated evolution of modular gene clusters. Nat Commun 2017; 8:1206. [PMID: 29089518 PMCID: PMC5663706 DOI: 10.1038/s41467-017-01344-3] [Citation(s) in RCA: 59] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2017] [Accepted: 09/08/2017] [Indexed: 11/09/2022] Open
Abstract
Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes. Reengineering polyketide synthase encoding genes to produce analogues of natural products can be slow and low-yielding. Here the authors use accelerated evolution to recombine the gene cluster for rapid production of rapamycin-related products.
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Affiliation(s)
- Aleksandra Wlodek
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Steve G Kendrew
- Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Engineered Biodesign Limited, Cambridge, CB22 3GN, UK
| | - Nigel J Coates
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Adam Hold
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Joanna Pogwizd
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Steven Rudder
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Lesley S Sheehan
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | | | - Anna E Stanley-Smith
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Tony Warneck
- Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Mohammad Nur-E-Alam
- Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, 12372, Saudi Arabia
| | - Markus Radzom
- Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,BASF SE, Speyerer Str. 2, Limburgerhof, 67117, Germany
| | - Christine J Martin
- Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Lois Overvoorde
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Markiyan Samborskyy
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK
| | - Silke Alt
- Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
| | - Daniel Heine
- Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK
| | - Guy T Carter
- Chemical and Screening Sciences, Wyeth Pharmaceuticals, 401 North Middletown Road, Pearl River, NY, 10965, USA
| | - Edmund I Graziani
- Chemical and Screening Sciences, Wyeth Pharmaceuticals, 401 North Middletown Road, Pearl River, NY, 10965, USA.,Medicine Discovery Network-Synthetic Biology, Pfizer Worldwide R&D, 445 Eastern Point Rd., Groton, CT, 06340, USA
| | - Frank E Koehn
- Chemical and Screening Sciences, Wyeth Pharmaceuticals, 401 North Middletown Road, Pearl River, NY, 10965, USA
| | - Leonard McDonald
- Chemical and Screening Sciences, Wyeth Pharmaceuticals, 401 North Middletown Road, Pearl River, NY, 10965, USA
| | - Alexander Alanine
- Roche Innovation Center Basel, Pharmaceutical Research and Early Development (PRED), Basel, CH-4070, Switzerland
| | | | - Suzan Keen Chao
- Roche Innovation Center Basel, Pharmaceutical Research and Early Development (PRED), Basel, CH-4070, Switzerland
| | - Hasane Ratni
- Roche Innovation Center Basel, Pharmaceutical Research and Early Development (PRED), Basel, CH-4070, Switzerland
| | - Lucinda Steward
- Roche Innovation Center Basel, Pharmaceutical Research and Early Development (PRED), Basel, CH-4070, Switzerland
| | - Isobel H Norville
- Defence Science and Technology Laboratory, Porton Down, PO17 6AD, UK
| | - Mitali Sarkar-Tyson
- Defence Science and Technology Laboratory, Porton Down, PO17 6AD, UK.,Marshall Centre for Infectious Diseases, School of Biomedical Sciences, University of Western Australia, Monash Avenue, Nedlands, WA, 6009, Australia
| | - Steven J Moss
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK
| | - Peter F Leadlay
- Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK
| | - Barrie Wilkinson
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK. .,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK. .,Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
| | - Matthew A Gregory
- Isomerase Therapeutics Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK. .,Biotica Technology Ltd., Chesterford Research Park, Cambridge, CB10 1XL, UK.
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32
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Nakazaki Y, Tsuyama T, Azuma Y, Takahashi M, Tada S. Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts. Biochem Biophys Res Commun 2017; 490:1375-1380. [PMID: 28694193 DOI: 10.1016/j.bbrc.2017.07.034] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2017] [Accepted: 07/07/2017] [Indexed: 10/19/2022]
Abstract
The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.
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Affiliation(s)
- Yuta Nakazaki
- Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo 164-8530, Japan
| | - Takashi Tsuyama
- Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan
| | - Yutaro Azuma
- Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan
| | - Mikiko Takahashi
- Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo 164-8530, Japan
| | - Shusuke Tada
- Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan.
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33
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Yang Y, Gao Y, Mutter-Rottmayer L, Zlatanou A, Durando M, Ding W, Wyatt D, Ramsden D, Tanoue Y, Tateishi S, Vaziri C. DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress. J Cell Biol 2017; 216:3097-3115. [PMID: 28835467 PMCID: PMC5626543 DOI: 10.1083/jcb.201702006] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2017] [Revised: 06/27/2017] [Accepted: 07/21/2017] [Indexed: 12/30/2022] Open
Abstract
The elevated CDK2 activity of oncogene-expressing cells induces DNA replication stress. Yang et al. show that the DNA repair protein RAD18 facilitates damage-tolerant DNA synthesis via the DNA polymerase κ in cells with aberrantly high CDK2 activity, suggesting an important new role for RAD18 in sustaining neoplastic cell survival. The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18−/− and Polκ−/− cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18–Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.
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Affiliation(s)
- Yang Yang
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Yanzhe Gao
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Liz Mutter-Rottmayer
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Anastasia Zlatanou
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Michael Durando
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Weimin Ding
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.,Oncology Center, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - David Wyatt
- Lineberger Comprehensive Cancer Center, Curriculumin Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Dale Ramsden
- Lineberger Comprehensive Cancer Center, Curriculumin Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC.,Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC
| | - Yuki Tanoue
- Division of Cell Maintenance, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
| | - Satoshi Tateishi
- Division of Cell Maintenance, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan
| | - Cyrus Vaziri
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC .,Lineberger Comprehensive Cancer Center, Curriculumin Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC
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34
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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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35
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Blumenfeld B, Ben-Zimra M, Simon I. Perturbations in the Replication Program Contribute to Genomic Instability in Cancer. Int J Mol Sci 2017; 18:E1138. [PMID: 28587102 PMCID: PMC5485962 DOI: 10.3390/ijms18061138] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2017] [Revised: 05/08/2017] [Accepted: 05/21/2017] [Indexed: 12/14/2022] Open
Abstract
Cancer and genomic instability are highly impacted by the deoxyribonucleic acid (DNA) replication program. Inaccuracies in DNA replication lead to the increased acquisition of mutations and structural variations. These inaccuracies mainly stem from loss of DNA fidelity due to replication stress or due to aberrations in the temporal organization of the replication process. Here we review the mechanisms and impact of these major sources of error to the replication program.
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Affiliation(s)
- Britny Blumenfeld
- Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
| | - Micha Ben-Zimra
- Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
- Pharmacology and Experimental Therapeutics Unit, The Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
| | - Itamar Simon
- Department of Microbiology and Molecular Genetics, IMRIC, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 91120, Israel.
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36
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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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37
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Multiple mechanisms contribute to double-strand break repair at rereplication forks in Drosophila follicle cells. Proc Natl Acad Sci U S A 2016; 113:13809-13814. [PMID: 27849606 DOI: 10.1073/pnas.1617110113] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Rereplication generates double-strand breaks (DSBs) at sites of fork collisions and causes genomic damage, including repeat instability and chromosomal aberrations. However, the primary mechanism used to repair rereplication DSBs varies across different experimental systems. In Drosophila follicle cells, developmentally regulated rereplication is used to amplify six genomic regions, two of which contain genes encoding eggshell proteins. We have exploited this system to test the roles of several DSB repair pathways during rereplication, using fork progression as a readout for DSB repair efficiency. Here we show that a null mutation in the microhomology-mediated end-joining (MMEJ) component, polymerase θ/mutagen-sensitive 308 (mus308), exhibits a sporadic thin eggshell phenotype and reduced chorion gene expression. Unlike other thin eggshell mutants, mus308 displays normal origin firing but reduced fork progression at two regions of rereplication. We also find that MMEJ compensates for loss of nonhomologous end joining to repair rereplication DSBs in a site-specific manner. Conversely, we show that fork progression is enhanced in the absence of both Drosophila Rad51 homologs, spindle-A and spindle-B, revealing homologous recombination is active and actually impairs fork movement during follicle cell rereplication. These results demonstrate that several DSB repair pathways are used during rereplication in the follicle cells and their contribution to productive fork progression is influenced by genomic position and repair pathway competition. Furthermore, our findings illustrate that specific rereplication DSB repair pathways can have major effects on cellular physiology, dependent upon genomic context.
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38
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Kotsantis P, Silva LM, Irmscher S, Jones RM, Folkes L, Gromak N, Petermann E. Increased global transcription activity as a mechanism of replication stress in cancer. Nat Commun 2016; 7:13087. [PMID: 27725641 PMCID: PMC5062618 DOI: 10.1038/ncomms13087] [Citation(s) in RCA: 234] [Impact Index Per Article: 26.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2016] [Accepted: 08/31/2016] [Indexed: 12/28/2022] Open
Abstract
Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer. Cancer cells proliferate at high rates and incur replication stress. Here, the authors show that this can be the consequence of oncogene-induced higher transcriptional activity, which, through increased RNA synthesis and R-loop accumulation, results in replication fork slowing and DNA damage.
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Affiliation(s)
- Panagiotis Kotsantis
- Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
| | - Lara Marques Silva
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Sarah Irmscher
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Rebecca M Jones
- Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
| | - Lisa Folkes
- Department of Oncology, CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK
| | - Natalia Gromak
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Eva Petermann
- Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
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Abstract
As the ratio of the copy number of the most replicated to the unreplicated regions in the same chromosome, the definition of chromosomal replication complexity (CRC) appears to leave little room for variation, being either two during S-phase or one otherwise. However, bacteria dividing faster than they replicate their chromosome spike CRC to four and even eight. A recent experimental inquiry about the limits of CRC in Escherichia coli revealed two major reasons to avoid elevating it further: (i) increased chromosomal fragmentation and (ii) complications with subsequent double-strand break repair. Remarkably, examples of stable elevated CRC in eukaryotic chromosomes are well known under various terms like "differential replication," "underreplication," "DNA puffs," "onion-skin replication," or "re-replication" and highlight the phenomenon of static replication fork (sRF). To accurately describe the resulting "amplification by overinitiation," I propose a new term: "replification" (subchromosomal overreplication). In both prokaryotes and eukaryotes, replification, via sRF processing, causes double-strand DNA breaks and, with their repair elevating chromosomal rearrangements, represents a novel genome instability factor. I suggest how static replication bubbles could be stabilized and speculate that some tandem duplications represent such persistent static bubbles. Moreover, I propose how static replication bubbles could be transformed into tandem duplications, double minutes, or inverted triplications. Possible experimental tests of these models are discussed.
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Affiliation(s)
- Andrei Kuzminov
- Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America
- * E-mail:
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40
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Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells. J Virol 2016; 90:6071-6084. [PMID: 27099318 DOI: 10.1128/jvi.00513-16] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2016] [Accepted: 04/16/2016] [Indexed: 01/04/2023] Open
Abstract
UNLABELLED The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.
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Petrakis TG, Komseli ES, Papaioannou M, Vougas K, Polyzos A, Myrianthopoulos V, Mikros E, Trougakos IP, Thanos D, Branzei D, Townsend P, Gorgoulis VG. Exploring and exploiting the systemic effects of deregulated replication licensing. Semin Cancer Biol 2016; 37-38:3-15. [PMID: 26707000 DOI: 10.1016/j.semcancer.2015.12.002] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2015] [Revised: 12/10/2015] [Accepted: 12/15/2015] [Indexed: 02/07/2023]
Abstract
Maintenance and accurate propagation of the genetic material are key features for physiological development and wellbeing. The replication licensing machinery is crucial for replication precision as it ensures that replication takes place once per cell cycle. Thus, the expression status of the components comprising the replication licensing apparatus is tightly regulated to avoid re-replication; a form of replication stress that leads to genomic instability, a hallmark of cancer. In the present review we discuss the mechanistic basis of replication licensing deregulation, which leads to systemic effects, exemplified by its role in carcinogenesis and a variety of genetic syndromes. In addition, new insights demonstrate that above a particular threshold, the replication licensing factor Cdc6 acts as global transcriptional regulator, outlining new lines of exploration. The role of the putative replication licensing factor ChlR1/DDX11, mutated in the Warsaw Breakage Syndrome, in cancer is also considered. Finally, future perspectives focused on the potential therapeutic advantage by targeting replication licensing factors, and particularly Cdc6, are discussed.
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Affiliation(s)
- Theodoros G Petrakis
- Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece
| | - Eirini-Stavroula Komseli
- Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece
| | - Marilena Papaioannou
- Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece
| | - Kostas Vougas
- Biomedical Research Foundation of the Academy of Athens, Athens, Greece
| | | | | | - Emmanuel Mikros
- Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Athens, Greece
| | - Ioannis P Trougakos
- Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Athens, Greece
| | - Dimitris Thanos
- Biomedical Research Foundation of the Academy of Athens, Athens, Greece
| | - Dana Branzei
- FIRC (Fondazione Italiana per la Ricerca sul Cancro) Institute of Molecular Oncology (IFOM), Milan, Italy
| | - Paul Townsend
- Faculty Institute of Cancer Sciences, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK
| | - Vassilis G Gorgoulis
- Molecular Carcinogenesis Group, Department of Histology and Embryology, School of Medicine, University of Athens, Athens, Greece; Biomedical Research Foundation of the Academy of Athens, Athens, Greece; Faculty Institute of Cancer Sciences, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
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42
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Franz A, Ackermann L, Hoppe T. Ring of Change: CDC48/p97 Drives Protein Dynamics at Chromatin. Front Genet 2016; 7:73. [PMID: 27200082 PMCID: PMC4853748 DOI: 10.3389/fgene.2016.00073] [Citation(s) in RCA: 67] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2016] [Accepted: 04/16/2016] [Indexed: 12/31/2022] Open
Abstract
The dynamic composition of proteins associated with nuclear DNA is a fundamental property of chromosome biology. In the chromatin compartment dedicated protein complexes govern the accurate synthesis and repair of the genomic information and define the state of DNA compaction in vital cellular processes such as chromosome segregation or transcription. Unscheduled or faulty association of protein complexes with DNA has detrimental consequences on genome integrity. Consequently, the association of protein complexes with DNA is remarkably dynamic and can respond rapidly to cellular signaling events, which requires tight spatiotemporal control. In this context, the ring-like AAA+ ATPase CDC48/p97 emerges as a key regulator of protein complexes that are marked with ubiquitin or SUMO. Mechanistically, CDC48/p97 functions as a segregase facilitating the extraction of substrate proteins from the chromatin. As such, CDC48/p97 drives molecular reactions either by directed disassembly or rearrangement of chromatin-bound protein complexes. The importance of this mechanism is reflected by human pathologies linked to p97 mutations, including neurodegenerative disorders, oncogenesis, and premature aging. This review focuses on the recent insights into molecular mechanisms that determine CDC48/p97 function in the chromatin environment, which is particularly relevant for cancer and aging research.
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Affiliation(s)
- André Franz
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Institute for Genetics, University of Cologne Cologne, Germany
| | - Leena Ackermann
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Institute for Genetics, University of Cologne Cologne, Germany
| | - Thorsten Hoppe
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, Institute for Genetics, University of Cologne Cologne, Germany
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43
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Popova T, Manié E, Boeva V, Battistella A, Goundiam O, Smith NK, Mueller CR, Raynal V, Mariani O, Sastre-Garau X, Stern MH. Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications. Cancer Res 2016; 76:1882-91. [PMID: 26787835 DOI: 10.1158/0008-5472.can-15-2128] [Citation(s) in RCA: 97] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2015] [Accepted: 01/08/2016] [Indexed: 11/16/2022]
Abstract
CDK12 is a recurrently mutated gene in serous ovarian carcinoma, whose downregulation is associated with impaired expression of DNA damage repair genes and subsequent hypersensitivity to DNA-damaging agents and PARP1/2 inhibitors. In this study, we investigated the genomic landscape associated with CDK12 inactivation in patients with serous ovarian carcinoma. We show that CDK12 loss was consistently associated with a particular genomic instability pattern characterized by hundreds of tandem duplications of up to 10 megabases (Mb) in size. Tandem duplications were characterized by a bimodal (∼0.3 and ∼3 Mb) size distribution and overlapping microhomology at the breakpoints. This genomic instability, denoted as the CDK12 TD-plus phenotype, is remarkably distinct from other alteration patterns described in breast and ovarian cancers. The CDK12 TD-plus phenotype was associated with a greater than 10% gain in genomic content and occurred at a 3% to 4% rate in The Cancer Genome Atlas-derived and in-house cohorts of patients with serous ovarian carcinoma. Moreover, CDK12-inactivating mutations together with the TD-plus phenotype were also observed in prostate cancers. Our finding provides new insight toward deciphering the function of CDK12 in genome maintenance and oncogenesis. Cancer Res; 76(7); 1882-91. ©2016 AACR.
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Affiliation(s)
- Tatiana Popova
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France.
| | - Elodie Manié
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France
| | - Valentina Boeva
- Institut Curie, Centre de Recherche, Paris, France. PSL Research University, Paris, France. INSERM U900, Paris, France
| | - Aude Battistella
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France
| | - Oumou Goundiam
- Institut Curie, Centre de Recherche, Paris, France. PSL Research University, Paris, France. EA4340-BCOH, Versailles Saint-Quentin-en-Yvelines University, Guyancourt, France. Institut Curie, Département de Biopathologie, Paris, France. Institut Curie, Département de Recherche Translationnelle, Paris, France
| | - Nicholas K Smith
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France
| | | | - Virginie Raynal
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France
| | - Odette Mariani
- PSL Research University, Paris, France. Institut Curie, Département de Biopathologie, Paris, France. Institut Curie, Centre de Ressources Biologiques, Paris, France
| | - Xavier Sastre-Garau
- PSL Research University, Paris, France. EA4340-BCOH, Versailles Saint-Quentin-en-Yvelines University, Guyancourt, France. Institut Curie, Département de Biopathologie, Paris, France
| | - Marc-Henri Stern
- Institut Curie, Centre de Recherche, Paris, France. INSERM U830, Paris, France. PSL Research University, Paris, France
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44
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Huang C, Cheng J, Bawa-Khalfe T, Yao X, Chin YE, Yeh ETH. SUMOylated ORC2 Recruits a Histone Demethylase to Regulate Centromeric Histone Modification and Genomic Stability. Cell Rep 2016; 15:147-157. [PMID: 27052177 DOI: 10.1016/j.celrep.2016.02.091] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Revised: 12/17/2015] [Accepted: 02/26/2016] [Indexed: 01/25/2023] Open
Abstract
Origin recognition complex 2 (ORC2), a subunit of the ORC, is essential for DNA replication initiation in eukaryotic cells. In addition to a role in DNA replication initiation at the G1/S phase, ORC2 has been shown to localize to the centromere during the G2/M phase. Here, we show that ORC2 is modified by small ubiquitin-like modifier 2 (SUMO2), but not SUMO1, at the G2/M phase of the cell cycle. SUMO2-modification of ORC2 is important for the recruitment of KDM5A in order to convert H3K4me3 to H3K4me2, a "permissive" histone marker for α-satellite transcription at the centromere. Persistent expression of SUMO-less ORC2 led to reduced α-satellite transcription and impaired pericentric heterochromatin silencing, which resulted in re-replication of heterochromatin DNA. DNA re-replication eventually activated the DNA damage response, causing the bypass of mitosis and the formation of polyploid cells. Thus, ORC2 sustains genomic stability by recruiting KDM5A to maintain centromere histone methylation in order to prevent DNA re-replication.
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Affiliation(s)
- Chao Huang
- Texas Heart Institute, Houston, TX 77030, USA; Department of Cardiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA; The Central Lab at Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Jinke Cheng
- Texas Heart Institute, Houston, TX 77030, USA; Department of Biochemistry and Molecular and Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Tasneem Bawa-Khalfe
- Department of Cardiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA; Center for Nuclear Receptors and Cell Signaling and Department of Biology and Biochemistry, University of Houston, Houston, TX 77204, USA
| | - Xuebiao Yao
- Anhui Key Laboratory of Cellular Dynamics and Hefei National Laboratory for Physical Sciences at Nanoscale, University of Science and Technology of China, Hefei 230026, China
| | - Y Eugene Chin
- The Central Lab at Shanghai Chest Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Edward T H Yeh
- Texas Heart Institute, Houston, TX 77030, USA; Department of Cardiology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA.
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45
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Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts. Biochem Biophys Res Commun 2016; 470:405-410. [DOI: 10.1016/j.bbrc.2016.01.028] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2016] [Accepted: 01/06/2016] [Indexed: 11/22/2022]
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46
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Paiva C, Godbersen JC, Berger A, Brown JR, Danilov AV. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents. Cell Death Dis 2015; 6:e1807. [PMID: 26158513 PMCID: PMC4650717 DOI: 10.1038/cddis.2015.161] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2015] [Revised: 05/06/2015] [Accepted: 05/12/2015] [Indexed: 02/06/2023]
Abstract
Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.
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Affiliation(s)
- C Paiva
- Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA
| | - J C Godbersen
- Geisel School of Medicine at Dartmouth, Hanover, NH, USA
| | - A Berger
- Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd, Cambridge, MA, USA
| | - J R Brown
- Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
| | - A V Danilov
- Knight Cancer Institute, Oregon Health and Science University, Portland, OR, USA
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47
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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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48
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Iglesias-Ara A, Zubiaga AM. The stress of coping with E2F loss. Mol Cell Oncol 2015; 3:e1038423. [PMID: 27308555 PMCID: PMC4845195 DOI: 10.1080/23723556.2015.1038423] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2015] [Revised: 04/01/2015] [Accepted: 04/02/2015] [Indexed: 11/10/2022]
Abstract
E2F transcription factors are key regulators of cellular proliferation, and altered E2F activity is a common feature of tumor cells. Thus, E2F targeting is being explored as a therapeutic strategy in cancer. Importantly, recent mouse knockout studies show that concomitant loss of E2f1/E2f2 activity is associated with increased genomic instability and oncogenic potential in normal differentiating cells, a finding that might have implications for cancer therapy.
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Affiliation(s)
- Ainhoa Iglesias-Ara
- Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, UPV/EHU , Bilbao, Spain
| | - Ana M Zubiaga
- Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country, UPV/EHU , Bilbao, Spain
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49
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Abstract
Genome instability is a hallmark of cancer, and DNA replication is the most vulnerable cellular process that can lead to it. Any condition leading to high levels of DNA damage will result in replication stress, which is a source of genome instability and a feature of pre-cancerous and cancerous cells. Therefore, understanding the molecular basis of replication stress is crucial to the understanding of tumorigenesis. Although a negative aspect of replication stress is its prominent role in tumorigenesis, a positive aspect is that it provides a potential target for cancer therapy. In this Review, we discuss the link between persistent replication stress and tumorigenesis, with the goal of shedding light on the mechanisms underlying the initiation of an oncogenic process, which should open up new possibilities for cancer diagnostics and treatment.
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Affiliation(s)
- Hélène Gaillard
- Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Av. Américo Vespucio s/n, Sevilla 41092, Spain
| | - Tatiana García-Muse
- Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Av. Américo Vespucio s/n, Sevilla 41092, Spain
| | - Andrés Aguilera
- Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Av. Américo Vespucio s/n, Sevilla 41092, Spain
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50
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Fu H, Martin MM, Regairaz M, Huang L, You Y, Lin CM, Ryan M, Kim R, Shimura T, Pommier Y, Aladjem MI. The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage. Nat Commun 2015; 6:6746. [PMID: 25879486 PMCID: PMC4400873 DOI: 10.1038/ncomms7746] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2014] [Accepted: 02/24/2015] [Indexed: 12/31/2022] Open
Abstract
The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.
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Affiliation(s)
- Haiqing Fu
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Melvenia M. Martin
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Marie Regairaz
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Liang Huang
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Yang You
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Chi-Mei Lin
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Michael Ryan
- InSilico Solutions, 11781 Lee Jackson Highway, Fairfax, VA 22033, USA
| | - RyangGuk Kim
- InSilico Solutions, 11781 Lee Jackson Highway, Fairfax, VA 22033, USA
| | - Tsutomu Shimura
- Department of Environmental Health, National Institute of Public Health 2-3-6 Minami, Wako, Saitama 351-0197, Japan
| | - Yves Pommier
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
| | - Mirit I. Aladjem
- Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
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