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

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
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.

scholarly journals Werner helicase control of human papillomavirus 16 E1-E2 DNA replication is regulated by SIRT1 deacetylation

2018 ◽  
Author(s):  
Dipon Das ◽  
Molly L Bristol ◽  
Nathan W Smith ◽  
Xu Wang ◽  
Pietro Pichierri ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Life Cycle ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Mammalian Cells ◽  
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 replication (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 where SIRT1 deacetylation of WRN promotes its interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication.

scholarly journals Inhibition of Karyopherin-α2 Augments Radiation-Induced Cell Death by Perturbing BRCA1-Mediated DNA Repair

2019 ◽  
Vol 20 (11) ◽  
pp. 2843 ◽  
Author(s):  
Kyung-Hee Song ◽  
Seung-Youn Jung ◽  
Jeong-In Park ◽  
Jiyeon Ahn ◽  
Jong Kuk Park ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Dna Repair ◽  
Apoptotic Cell ◽  
Nucleocytoplasmic Transport ◽  
Repair Protein ◽  
Damage Response ◽  
Dna Repair Protein ◽  
Radiation Induced ◽  
Early Local Recurrence

Ionizing radiation (IR) has been widely used in the treatment of cancer. Radiation-induced DNA damage triggers the DNA damage response (DDR), which can confer radioresistance and early local recurrence by activating DNA repair pathways. Since karyopherin-α2 (KPNA2), playing an important role in nucleocytoplasmic transport, was significantly increased by IR in our previous study, we aimed to determine the function of KPNA2 with regard to DDR. Exposure to radiation upregulated KPNA2 expression in human colorectal cancer HT29 and HCT116 cells and breast carcinoma MDA-MB-231 cells together with the increased expression of DNA repair protein BRCA1. The knockdown of KPNA2 effectively increased apoptotic cell death via inhibition of BRCA1 nuclear import following IR. Therefore, we propose that KPNA2 is a potential target for overcoming radioresistance via interruption to DDR.

scholarly journals BET inhibition induces HEXIM1- and RAD51-dependent conflicts between transcription and replication

2018 ◽  
Author(s):  
Akhil Bowry ◽  
Ann Liza Piberger ◽  
Eva Petermann
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Dna Damage Response ◽  
Bet Inhibitor ◽  
Bet Inhibitors ◽  
Damage Response ◽  
Bromodomain Proteins ◽  
Brd4 Inhibition

SUMMARYBET bromodomain proteins are epigenetic readers required for oncogenic transcription activities, and BET inhibitors have been rapidly advanced into clinical trials. Understanding the effects of BET inhibition on other nuclear processes such as DNA replication will be important for future clinical applications. Here we show that BET inhibition causes replication stress in cancer and non-cancer cells due to a rapid burst in global RNA synthesis and interference of transcription with replication. We identify BRD4 as the main BET inhibitor target in this process and provide evidence that BRD4 inhibition causes transcription-replication interference through release of P-TEFb from its inhibitor HEXIM1, promoting RNA Polymerase II phosphorylation. Unusually, BET inhibitor-induced transcription-replication interference does not activate the classic ATM/ATR-dependent DNA damage response. We show however that they promote foci formation of the homologous recombination factor RAD51. Both HEXIM1 and RAD51 are required for BET inhibitor-induced fork slowing, but rescuing fork slowing by HEXIM1 or RAD51 depletion activate a DNA damage response. Our data support a new mechanism where BRD4 inhibition slows replication and suppresses DNA damage through concerted action of transcription and homologous recombination machineries. They shed new light on the roles of DNA replication and recombination in the action of this new class of cancer drugs.

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

2015 ◽  
Vol 90 (5) ◽  
pp. 2639-2652 ◽  
Author(s):  
William H. Chappell ◽  
Dipendra Gautam ◽  
Suzan T. Ok ◽  
Bryan A. Johnson ◽  
Daniel C. Anacker ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Human Papillomavirus ◽  
Homologous Recombination ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Productive Replication

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.

scholarly journals FANCD2 Binds Human Papillomavirus Genomes and Associates with a Distinct Set of DNA Repair Proteins to Regulate Viral Replication

mBio ◽  
2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Chelsey C. Spriggs ◽  
Laimonis A. Laimins
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Human Papillomavirus ◽  
Life Cycle ◽  
High Risk ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Repair Pathway ◽  
Damage Response ◽  
Dna Repair Proteins

ABSTRACTThe life cycle of human papillomavirus (HPV) is dependent on the differentiation state of its host cell. HPV genomes are maintained as low-copy episomes in basal epithelial cells and amplified to thousands of copies per cell in differentiated layers. Replication of high-risk HPVs requires the activation of the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) DNA repair pathways. The Fanconi anemia (FA) pathway is a part of the DNA damage response and mediates cross talk between the ATM and ATR pathways. Our studies show that HPV activates the FA pathway, leading to the accumulation of a key regulatory protein, FANCD2, in large nuclear foci. These HPV-dependent foci colocalize with a distinct population of DNA repair proteins, including ATM components γH2AX and BRCA1, but infrequently with p-SMC1, which is required for viral genome amplification in differentiated cells. Furthermore, FANCD2 is found at viral replication foci, where it is preferentially recruited to viral genomes compared to cellular chromosomes and is required for maintenance of HPV episomes in undifferentiated cells. These findings identify FANCD2 as an important regulator of HPV replication and provide insight into the role of the DNA damage response in the differentiation-dependent life cycle of HPV.IMPORTANCEHigh-risk human papillomaviruses (HPVs) are the etiological agents of cervical cancer and are linked to the development of many other anogenital and oropharyngeal cancers. Identification of host cellular pathways involved in regulating the viral life cycle may be helpful in identifying treatments for HPV lesions. Mutations in genes of the Fanconi anemia (FA) DNA repair pathway lead to genomic instability in patients and a predisposition to HPV-associated malignancies. Our studies demonstrate that FA pathway component FANCD2 is recruited to HPV DNA, associates with members of the ATM DNA repair pathway, and is essential for the maintenance of viral episomes in basal epithelial cells. Disruption of the FA pathway may result in increased integration events and a higher incidence of HPV-related cancer. Our study identifies new links between HPV and the FA pathway that may help to identify new therapeutic targets for the treatment of existing HPV infections and cancers.

scholarly journals Werner syndrome protein (WRN) regulates cell proliferation and the human papillomavirus 16 life cycle during epithelial differentiation

2020 ◽  
Author(s):  
Claire D. James ◽  
Dipon Das ◽  
Ethan L. Morgan ◽  
Raymonde Otoa ◽  
Andrew Macdonald ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Life Cycle ◽  
Dna Replication ◽  
Viral Replication ◽  
Dna Damage Response ◽  
Basal Cell ◽  
Viral Life Cycle ◽  
Cervical Lesions ◽  
Damage Response

AbstractHuman papillomaviruses recruit a host of DNA damage response factors to their viral genome to facilitate homologous recombination replication in association with the viral replication factors E1 and E2. We previously demonstrated that SIRT1 deacetylation of WRN promotes recruitment of WRN to E1-E2 replicating DNA, and that WRN regulates both the levels and fidelity of E1-E2 replication. The deacetylation of WRN by SIRT1 results in an active protein able to complex with replicating DNA, but a protein that is less stable. Here we demonstrate an inverse correlation between SIRT1 and WRN in CIN cervical lesions when compared with normal control tissue, supporting our model of SIRT1 deacetylation destabilizing WRN protein. We CRISPR/Cas9 edited N/Tert-1 and N/Tert-1+HPV16 cells to knock out WRN protein expression and subjected the cells to organotypic raft cultures. In N/Tert-1 cells without WRN expression there was enhanced basal cell proliferation, DNA damage and thickening of the differentiated epithelium. In N/Tert-1+HPV16 cells, there was enhanced basal cell proliferation, increased DNA damage throughout the epithelium and increased viral DNA replication. Overall, the results demonstrate that the expression of WRN is required to control the proliferation of N/Tert-1 cells and controls the HPV16 life cycle in these cells. This complements our previous data demonstrating that WRN controls the levels and fidelity of HPV16 E1-E2 DNA replication. The results describe a new role for WRN, a tumor suppressor, in controlling keratinocyte differentiation and the HPV16 life cycle.ImportanceHPV16 is the major human viral carcinogen, responsible for around 3-4% of all cancers worldwide. Our understanding of how the viral replication machinery interacts with host factors to control/activate the DNA damage response to promote the viral life cycle remains incomplete. Recently, we demonstrated a SIRT1-WRN axis that controls HPV16 replication and here we demonstrate that this axis persists in clinical cervical lesions induced by HPV16. Here we describe the effects of WRN depletion on cellular differentiation with and without HPV16; WRN depletion results in enhanced proliferation and DNA damage irrespective of HPV16 status. Also, WRN is a restriction factor for the viral life cycle as replication is disrupted in the absence of WRN. Future studies will focus on enhancing our understanding of how WRN regulates viral replication. Our goal is to ultimately identify cellular factors essential for HPV16 replication that can be targeted for therapeutic gain.

Increased γH2AX Foci in Old Hematopoietic Stem Cells Are Independent of the DNA Damage Response and Linked to Inefficient DNA Replication/Transcription

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1207-1207
Author(s):  
Johanna Flach ◽  
Sietske Bakker ◽  
Pauline Conroy ◽  
Damien Reynaud ◽  
Michelle M. Le Beau ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Dna Double Strand Breaks ◽  
Hematopoietic Stem ◽  
Strand Breaks ◽  
Γh2ax Foci ◽  
Damage Response

Abstract Abstract 1207 Hematopoietic stem cells (HSCs) are responsible for the life-long production of all blood cells. Changes in the biological function of old HSCs have been directly linked to the occurrence of age-related blood defects including immunosenescence, anemia and the development of a broad spectrum of hematological disorders (i.e., myeloproliferative neoplasms, leukemia, bone marrow failure). Gene expression studies and analysis of genetically modified mice have also suggested that error-prone DNA repair as well as a decrease in genomic stability are one of the driving forces for the reduced functional capacity of old HSCs. Here, we used HSCs (Lin-/c-Kit+/Sca-1+/Flk2-/CD48-/CD150+) isolated from the bone marrow of old (20–24 months old) and young (6–12 weeks old) C57Bl/6 mice to directly investigate the DNA damage response of old HSCs. Using immunofluorescence, we first confirmed that freshly isolated quiescent old HSCs have an increased number of γH2AX foci, which is a well-established indicator for DNA double-strand breaks. In addition, we found that these intrinsically occurring γH2AX foci specifically co-localized with nucleolar markers (i.e., UBF, fibrillarin and nucleolin) and were a cell-intrinsic feature of old HSCs as demonstrated by transplantation experiments of old HSCs into young recipient mice. However, we could not demonstrate that nucleolar γH2AX foci in old HSCs represent sites of DNA damage. Neither did known DNA repair-associated markers like 53BP1 co-localize with nucleolar γH2AX foci, nor were other DNA damage markers such as phospho-ATM or PARP1 increased in old HSCs. In addition, none of the other methods we used to measure DNA fragmentation such as TUNEL or COMET assays revealed elevated levels of DNA damage in old HSCs, and spectral karyotyping (SKY) analysis of in vitro cultured old HSCs did not provide evidence for DNA damage-associated chromosomal alterations. We then used 2Gy ionizing radiation (IR) to directly induce DNA double-strand breaks and measured the DNA repair capacity of old HSCs. Strikingly, we observed a similar DNA damage response and DNA repair kinetics in young and old HSCs. These results provide evidence that old HSCs can respond adequately to DNA damage and that accumulation of γH2AX at the nucleolus is not the consequence of an activated DNA damage response. The nucleolus consists of a highly regulated repetitive sequence of rDNA units and is the site of ribosomal DNA transcription. Both young and old quiescent HSCs have well-formed nucleoli as shown by electron microscopy analyses. Strikingly, we found a complete disappearance of nucleolar γH2AX foci when old HSCs are forced into cell cycle upon in vitro culture. Furthermore, we observed a significant delay in the onset of the first cell division and timing of nucleolar reformation following mitosis in old HSCs. In addition, cycling old HSCs displayed higher levels of DNA replication/transcription-associated γH2AX foci compared to cycling young HSCs. We are currently investigating how defects in the DNA replication machinery could contribute to the nucleolar γH2AX foci and cell cycle features of old HSCs, and whether old HSCs maintain similar levels of rRNA transcription as compared to young HSCs. Taken together, our results demonstrate that the increased numbers of γH2AX foci observed in old HSCs is not caused by an accumulation of DNA damage. Instead, nucleolar γH2AX foci appear to be a hallmark feature of quiescent old HSCs that could reflect epigenetic changes in rDNA chromatin structure linked to inefficient DNA replication/transcription. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activating the DNA Damage Response and Suppressing Innate Immunity: Human Papillomaviruses Walk the Line

Pathogens ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 467 ◽  
Author(s):  
Claire D. James ◽  
Dipon Das ◽  
Molly L. Bristol ◽  
Iain M. Morgan
Keyword(s):  
Immune Response ◽  
Dna Damage ◽  
Life Cycle ◽  
Dna Damage Response ◽  
Innate Immune Response ◽  
Adaptive Immune Response ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Innate Immune ◽  
Damage Response

Activation of the DNA damage response (DDR) by external agents can result in DNA fragments entering the cytoplasm and activating innate immune signaling pathways, including the stimulator of interferon genes (STING) pathway. The consequences of this activation can result in alterations in the cell cycle including the induction of cellular senescence, as well as boost the adaptive immune response following interferon production. Human papillomaviruses (HPV) are the causative agents in a host of human cancers including cervical and oropharyngeal; HPV are responsible for around 5% of all cancers. During infection, HPV replication activates the DDR in order to promote the viral life cycle. A striking feature of HPV-infected cells is their ability to continue to proliferate in the presence of an active DDR. Simultaneously, HPV suppress the innate immune response using a number of different mechanisms. The activation of the DDR and suppression of the innate immune response are essential for the progression of the viral life cycle. Here, we describe the mechanisms HPV use to turn on the DDR, while simultaneously suppressing the innate immune response. Pushing HPV from this fine line and tipping the balance towards activation of the innate immune response would be therapeutically beneficial.

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