Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

ABSTRACTHigh-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination.IMPORTANCEProductive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle.

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Recent advances in the nucleolar responses to DNA double-strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa713 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9449-9461

Author(s):

Lea Milling Korsholm ◽

Zita Gál ◽

Blanca Nieto ◽

Oliver Quevedo ◽

Stavroula Boukoura ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Ribosomal Dna ◽

Repetitive Sequences ◽

Dna Lesions ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

Abstract DNA damage poses a serious threat to human health and cells therefore continuously monitor and repair DNA lesions across the genome. Ribosomal DNA is a genomic domain that represents a particular challenge due to repetitive sequences, high transcriptional activity and its localization in the nucleolus, where the accessibility of DNA repair factors is limited. Recent discoveries have significantly extended our understanding of how cells respond to DNA double-strand breaks (DSBs) in the nucleolus, and new kinases and multiple down-stream targets have been identified. Restructuring of the nucleolus can occur as a consequence of DSBs and new data point to an active regulation of this process, challenging previous views. Furthermore, new insights into coordination of cell cycle phases and ribosomal DNA repair argue against existing concepts. In addition, the importance of nucleolar-DNA damage response (n-DDR) mechanisms for maintenance of genome stability and the potential of such factors as anti-cancer targets is becoming apparent. This review will provide a detailed discussion of recent findings and their implications for our understanding of the n-DDR. The n-DDR shares features with the DNA damage response (DDR) elsewhere in the genome but is also emerging as an independent response unique to ribosomal DNA and the nucleolus.

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SET8 Is a Potential Therapeutic Target in MM

Blood ◽

10.1182/blood.v128.22.4435.4435 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4435-4435

Author(s):

Herviou Laurie ◽

Fanny Izard ◽

Elke De Bruyne ◽

Eva Desmedt ◽

Anqi Ma ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Myeloma Cells ◽

Strand Breaks ◽

Damage Response

Abstract Epigenetic regulation mechanisms - such as histone marks, DNA methylation and miRNA - are often misregulated in cancers and are associated with tumorigenesis and drug resistance. Multiple Myeloma (MM) is a malignant plasma cell disease that accumulates within the bone marrow. Epigenetic modifications in MM are associated not only with cancer development and progression, but also with resistance to chemotherapy. This epigenetic plasticity can be targeted with epidrugs, nowadays used in treatment of several cancers. We recently identified a significant overexpression of the lysine histone methyltransferase SETD8 in MM cells (HMCLs; N=40) compared with normal plasma cells (N=5) (P<0.001). SETD8 (also known as SET8, PR-Set7, KMT5A) is the sole enzyme responsible for the monomethylation of histone H4 at lysine 20 (H4K20me1) which has been linked to chromatin compaction and cell-cycle regulation. In addition, SETD8 induces the methylation of non-histone proteins, such as the replication factor PCNA, the tumor suppressor P53 and its stabilizing protein Numb. While SETD8-mediated methylation of P53 and Numb inhibits apoptosis, PCNA methylation upon SETD8 enhances the interaction with the Flap endonuclease FEN1 and promotes cancer cell proliferation. SETD8 is also implicated in DNA damage response, helping 53BP1 recruitment at DNA double-strand breaks. Consistent with this, overexpression of SETD8 is found in various types of cancer and has been directly implicated in breast cancer invasiveness and metastasis. A role of SETD8 in development of MM has however never been described. We found that high SETD8 expression is associated with a poor prognosis in 2 independent cohorts of newly diagnosed patients (UAMS-TT2 cohort - N=345 and UAMS-TT3 cohort - N=158). Specific SETD8 inhibition with UNC-0379 inhibitor, causing its degradation and H4K20me1 depletion, leads to significant growth inhibition of HMCLs (N=10) and the murine cell lines 5T33MM and 5TGM1. MM cells treated with UNC-0379 presented a G0/G1 cell cycle arrest after 24h of treatment, followed by apoptosis 48h later. To confirm that SETD8 inhibition is as efficient on primary MM cells from patients, primary MM cells (N=8) were co-cultured with their bone marrow microenvironment and recombinant IL-6 and treated for 4 days with UNC-0379. Interestingly, treatment of MM patient samples with UNC-0379 reduces the percentage of myeloma cells (65%; P<0.005) without significantly affecting the non-myeloma cells, suggesting a specific addiction of primary myeloma cells to SETD8 activity. Melphalan is an alkylating agent commonly used in MM treatment. As SETD8 is known to be involved in the DNA damage response, we investigated the effect of its combination with Melphalan on HMCLs. Results show that this particular drug combination strongly enhances double strand breaks in HMCLs monitored using 53BP1 foci formation and gH2AX detection. This result emphasizes a potential role of SETD8 in DNA repair in MM cells. Furthermore, GSEA analysis of patients with high SETD8 expression highlighted a significant enrichment of genes involved in DNA repair, MYC-MAX targets and MAPK pathway. Our study is the first to demonstrate the importance of SETD8 for MM cells survival and suggest that SETD8 inhibition represent a promising strategy to improve conventional treatment of MM with DNA damaging agents. Disclosures No relevant conflicts of interest to declare.

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HIC1 (hypermethylated in cancer 1) SUMOylation is dispensable for DNA repair but is essential for the apoptotic DNA damage response (DDR) to irreparable DNA double-strand breaks (DSBs)

Oncotarget ◽

10.18632/oncotarget.13807 ◽

2016 ◽

Vol 8 (2) ◽

pp. 2916-2935 ◽

Cited By ~ 8

Author(s):

Sonia Paget ◽

Marion Dubuissez ◽

Vanessa Dehennaut ◽

Joe Nassour ◽

Brennan T. Harmon ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response

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Synthetic Lethality In CLL With DNA Damage Response Defect By Targeting ATR Pathway

Blood ◽

10.1182/blood.v122.21.120.120 ◽

2013 ◽

Vol 122 (21) ◽

pp. 120-120

Author(s):

Tatjana Stankovic ◽

Davies Nicholas ◽

Marwan Kwok ◽

Edward Smith ◽

Eliot Yates ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Synthetic Lethality ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Single Strand Break ◽

Strand Breaks ◽

Damage Response ◽

Repair Pathways

Abstract Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed. ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality’ and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM. First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea. We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support. To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss. We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures: Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

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The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

The Journal of Cell Biology ◽

10.1083/jcb.201001160 ◽

2010 ◽

Vol 191 (1) ◽

pp. 31-43 ◽

Cited By ~ 124

Author(s):

Ye Xu ◽

Yingli Sun ◽

Xiaofeng Jiang ◽

Marina K. Ayrapetov ◽

Patryk Moskwa ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Active Process ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response ◽

Significant Barrier ◽

Nucleosome Stability

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.

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High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair

AJP Renal Physiology ◽

10.1152/ajprenal.00060.2003 ◽

2003 ◽

Vol 285 (2) ◽

pp. F266-F274 ◽

Cited By ~ 44

Author(s):

Natalia I. Dmitrieva ◽

Dmitry V. Bulavin ◽

Maurice B. Burg

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Dna Repair ◽

Cell Cycle Arrest ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Cycle Arrest ◽

Damage Response

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2 phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.

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Sae2 antagonizes Rad9 accumulation at DNA double-strand breaks to attenuate checkpoint signaling and facilitate end resection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816539115 ◽

2018 ◽

Vol 115 (51) ◽

pp. E11961-E11969 ◽

Cited By ~ 14

Author(s):

Tai-Yuan Yu ◽

Michael T. Kimble ◽

Lorraine S. Symington

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Inhibitory Effects ◽

Checkpoint Signaling ◽

Synthetic Lethal ◽

Strand Breaks ◽

Damage Response ◽

End Resection

The Mre11-Rad50-Xrs2NBS1 complex plays important roles in the DNA damage response by activating the Tel1ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs). To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2CtIP. Accordingly, Mre11-nuclease deficient (mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes; however, we found several notable differences. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. Third, Sae2 attenuates the Tel1-Rad53CHK2 checkpoint and antagonizes Rad953BP1 accumulation at DSBs independent of Mre11 nuclease. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint.

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Werner Helicase Control of Human Papillomavirus 16 E1-E2 DNA Replication Is Regulated by SIRT1 Deacetylation

mBio ◽

10.1128/mbio.00263-19 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 7

Author(s):

Dipon Das ◽

Molly L. Bristol ◽

Nathan W. Smith ◽

Claire D. James ◽

Xu Wang ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Life Cycle ◽

Dna Replication ◽

Homologous Recombination ◽

Dna Damage Response ◽

Human Papillomaviruses ◽

Repair Protein ◽

Damage Response ◽

Dna Repair Protein

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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