Replication-Dependent DNA Damage Response Triggered by Roscovitine Induces an Uncoupling of DNA Replication Proteins

ABSTRACTHuman papillomaviruses (HPV) are double-stranded DNA viruses causative in a host of human diseases, including several cancers. Following infection, two viral proteins, E1 and E2, activate viral replication in association with cellular factors and stimulate the DNA damage response (DDR) during the replication process. E1-E2 uses homologous recombination (HR) to facilitate DNA replication, but an understanding of host factors involved in this process remains incomplete. Previously, we demonstrated that the class III deacetylase SIRT1, which can regulate HR, is recruited to E1-E2-replicating DNA and regulates the level of replication. Here, we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-E2-replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of WRN, but a reduced ability to interact with E1-E2-replicating DNA. We present a model in which E1-E2 replication turns on the DDR, stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN interaction with E1-E2-replicating DNA to control the quantity and fidelity of replication. As well as offering a crucial insight into HPV replication control, this system offers a unique model for investigating the link between SIRT1 and WRN in controlling replication in mammalian cells.IMPORTANCEHPV16 is the major viral human carcinogen responsible for between 3 and 4% of all cancers worldwide. Following infection, this virus activates the DNA damage response (DDR) to promote its life cycle and recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 replication interacts with the DDR remains incomplete. Here, we demonstrate that the cellular deacetylase SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 replication. Overall, the results suggest a mechanism by which SIRT1 deacetylation of WRN promotes its interaction with E1-E2-replicating DNA to control the levels and fidelity of that replication.

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Bocavirus Infection Induces a DNA Damage Response That Facilitates Viral DNA Replication and Mediates Cell Death

Journal of Virology ◽

10.1128/jvi.01534-10 ◽

2010 ◽

Vol 85 (1) ◽

pp. 133-145 ◽

Cited By ~ 36

Author(s):

Y. Luo ◽

A. Y. Chen ◽

J. Qiu

Keyword(s):

Dna Damage ◽

Cell Death ◽

Dna Replication ◽

Dna Damage Response ◽

Viral Dna ◽

Viral Dna Replication ◽

Damage Response

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Coordinated Chromatin-Association of Fanconi Anemia Network Proteins Requires Replication-Coupled DNA Damage Recognition.

Blood ◽

10.1182/blood.v104.11.723.723 ◽

2004 ◽

Vol 104 (11) ◽

pp. 723-723

Author(s):

Alexandra Sobeck ◽

Stacie Stone ◽

Bendert deGraaf ◽

Vincenzo Costanzo ◽

Johan deWinter ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Fanconi Anemia ◽

Bone Marrow Failure ◽

S Phase ◽

Dependent Manner ◽

Core Complex ◽

Damage Response ◽

Crosslinking Agents

Abstract Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA crosslinking agents and diverse clinical symptoms, including developmental anomalies, progressive bone marrow failure, and predisposition to leukemias and other cancers. FA is genetically heterogeneous, resulting from mutations in any of at least eleven different genes. The FA proteins function together in a pathway composed of a mulitprotein core complex that is required to trigger the DNA-damage dependent activation of the downstream FA protein, FANCD2. This activation is thought to be the key step in a DNA damage response that functionally links FA proteins to major breast cancer susceptibility proteins BRCA1 and BRCA2 (BRCA2 is FA gene FANCD1). The essential function of the FA proteins is unknown, but current models suggest that FA proteins function at the interface between cell cycle checkpoints, DNA repair and DNA replication, and are likely to play roles in the DNA damage response during S phase. To provide a platform for dissecting the key functional events during S-phase, we developed cell-free assays for FA proteins based on replicating extracts from Xenopus eggs. We identified the Xenopus homologs of human FANCD2 (xFANCD2) and several of the FA core complex proteins (xCCPs), and biochemically characterized these proteins in replicating cell-free extracts. We found that xCCPs and a modified isoform of xFANCD2 become associated with chromatin during normal and disrupted DNA replication. Blocking initiation of replication with geminin demonstrated that association of xCCPs and xFANCD2 with chromatin occurs in a strictly replication-dependent manner that is enhanced following DNA damage by crosslinking agents or by addition of aphidicolin, an inhibitor of replicative DNA polymerases. In addition, chromatin binding of xFANCD2, but not xBRCA2, is abrogated when xFANCA is quantitatively depleted from replicating extracts suggesting that xFANCA promotes the loading of xFANCD2 on chromatin. The chromatin-association of xFANCD2 and xCCPs is diminished in the presence of caffeine, an inhibitor of checkpoint kinases. Taken together, our data suggest a model in which the ordered loading of FA proteins on chromatin is required for processing a subset of DNA replication-blocking lesions that are resolved during late stages of replication.

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Kinetics of the UV-induced DNA damage response in relation to cell cycle phase. Correlation with DNA replication

Cytometry Part A ◽

10.1002/cyto.a.20839 ◽

2009 ◽

pp. n/a-n/a ◽

Cited By ~ 9

Author(s):

Hong Zhao ◽

Frank Traganos ◽

Zbigniew Darzynkiewicz

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Cell Cycle Phase ◽

Phase Correlation ◽

Cycle Phase ◽

Damage Response ◽

Kinetics Of

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Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1525620113 ◽

2016 ◽

Vol 113 (26) ◽

pp. E3676-E3685 ◽

Cited By ~ 8

Author(s):

Nicholas A. Willis ◽

Chunshui Zhou ◽

Andrew E. H. Elia ◽

Johanne M. Murray ◽

Antony M. Carr ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Dna Replication ◽

Fission Yeast ◽

Dna Damage Response ◽

Cellular Response ◽

Genome Stability ◽

Biological Significance ◽

S Phase ◽

Damage Response

The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase–specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

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Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage

Cytometry Part A ◽

10.1002/cyto.a.22325 ◽

2013 ◽

pp. n/a-n/a

Author(s):

Tytus Bernas ◽

Krzysztof Berniak ◽

Paulina Rybak ◽

Mirosław Zarębski ◽

Hong Zhao ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Oxidative Damage ◽

Dna Damage Response ◽

Replication Stress ◽

Spatial Correlations ◽

Damage Response

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Breaking the DNA Damage Response via Serine/Threonine Kinase Inhibitors to Improve Cancer Treatment

Current Medicinal Chemistry ◽

10.2174/0929867325666180117102233 ◽

2019 ◽

Vol 26 (8) ◽

pp. 1425-1445 ◽

Cited By ~ 3

Author(s):

Wioletta Rozpędek ◽

Dariusz Pytel ◽

Alicja Nowak-Zduńczyk ◽

Dawid Lewko ◽

Radosław Wojtczak ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Cancer Treatment ◽

Dna Damage Response ◽

Ataxia Telangiectasia ◽

Kinase Inhibitors ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Anti Cancer ◽

Damage Response

Multiple, both endogenous and exogenous, sources may induce DNA damage and DNA replication stress. Cells have developed DNA damage response (DDR) signaling pathways to maintain genomic stability and effectively detect and repair DNA lesions. Serine/ threonine kinases such as Ataxia-telangiectasia mutated (ATM) and Ataxia-telangiectasia and Rad3-Related (ATR) are the major regulators of DDR, since after sensing stalled DNA replication forks, DNA double- or single-strand breaks, may directly phosphorylate and activate their downstream targets, that play a key role in DNA repair, cell cycle arrest and apoptotic cell death. Interestingly, key components of DDR signaling networks may constitute an attractive target for anti-cancer therapy through two distinct potential approaches: as chemoand radiosensitizers to enhance the effectiveness of currently used genotoxic treatment or as single agents to exploit defects in DDR in cancer cells via synthetic lethal approach. Moreover, the newest data reported that serine/threonine protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is also closely associated with cancer development and progression. Thereby, utilization of small-molecule, serine/threonine kinase inhibitors may provide a novel, groundbreaking, anti-cancer treatment strategy. Currently, a range of potent, highlyselective toward ATM, ATR and PERK inhibitors has been discovered, but after foregoing study, additional investigations are necessary for their future clinical use.

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Functional dissection of proliferating-cell nuclear antigens (1 and 2) in human malarial parasite Plasmodium falciparum: possible involvement in DNA replication and DNA damage response

Biochemical Journal ◽

10.1042/bj20150452 ◽

2015 ◽

Vol 470 (1) ◽

pp. 115-129 ◽

Cited By ~ 15

Author(s):

Pallabi Mitra ◽

Khadija Banu ◽

Abhijit S. Deshmukh ◽

Naidu Subbarao ◽

Suman Kumar Dhar

Keyword(s):

Dna Damage ◽

Plasmodium Falciparum ◽

Dna Replication ◽

Dna Damage Response ◽

Malarial Parasite ◽

Nuclear Antigens ◽

Parasite Plasmodium ◽

Damage Response ◽

Parasite Plasmodium Falciparum ◽

Proliferating Cell

P. falciparum harbours two PCNAs. We show unambiguously that PfPCNA1 is the primary replicative PCNA out of the two PCNAs. Both the PfPCNAs are involved in the robust DNA-damage response that the parasite mounts in a genotoxic environment.

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Isolation of crt mutants constitutive for transcription of the DNA damage inducible gene RNR3 in Saccharomyces cerevisiae.

Genetics ◽

10.1093/genetics/131.4.851 ◽

1992 ◽

Vol 131 (4) ◽

pp. 851-866 ◽

Cited By ~ 9

Author(s):

Z Zhou ◽

S J Elledge

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Lacz Reporter ◽

Dna Damaging Agents ◽

Rate Limiting Step ◽

Damage Response ◽

Complementation Groups ◽

Essential Enzyme ◽

Inducible Genes

Abstract Ribonucleotide reductase is an essential enzyme that catalyzes the rate limiting step for production of the deoxyribonucleotides required for DNA synthesis. It is encoded by three genes, RNR1, RNR2 and RNR3, each of which is inducible by agents that damage DNA or block DNA replication. To probe the signaling pathway mediating this DNA damage response, we have designed a general selection system for isolating spontaneous trans-acting mutations that alter RNR3 expression using a chromosomal RNR3-URA3 transcriptional fusion and an RNR3-lacZ reporter plasmid. Using this system, we have isolated 202 independent trans-acting crt (constitutive RNR3 transcription) mutants that express high levels of RNR3 in the absence of DNA damaging agents. Of these, 200 are recessive and fall into 9 complementation groups. In some crt groups, the expression of RNR1 and RNR2 are also elevated, suggesting that all three RNR genes share a common regulatory pathway. Mutations in most CRT genes confer additional phenotypes, among these are clumpiness, hydroxyurea sensitivity, temperature sensitivity and slow growth. Five of the CRT genes have been identified as previously cloned genes; CRT4 is TUP1, CRT5 is POL1/CDC17, CRT6 is RNR2, CRT7 is RNR1, and CRT8 is SSN6. crt6-68 and crt7-240 are the first ts alleles of RNR2 and RNR1, respectively, and arrest with a large budded, cdc terminal phenotype at the nonpermissive temperature. The isolation of crt5-262, an additional cdc allele of POL1/CDC17, suggests for the first time that directly blocking DNA replication can provide a signal to induce the DNA damage response. crt2 mutants show a defect in basal level expression of RNR1-lacZ reporter constructs. These are the first mutants isolated in yeast that alter the regulation of DNA damage inducible genes and the identification of their functions sheds light on the DNA damage sensory network.

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