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

Abstract

16Human papillomaviruses (HPV) are double stranded DNA viruses causative in a host of human diseases 17 including several cancers. Following infection two viral proteins, E1 and E2, activate viral replication in 18 association with cellular factors, and stimulate the DNA damage response (DDR) during the replication 19 process. E1-E2 uses homologous replication (HR) to facilitate DNA replication, but an understanding of 20 host factors involved in this process remains incomplete. Previously we demonstrated that the class III 21 deacetylase SIRT1, which can regulate HR, is recruited to E1-E2 replicating DNA and regulates the level 22 of replication. Here we demonstrate that SIRT1 promotes the fidelity of E1-E2 replication and that the 23 absence of SIRT1 results in reduced recruitment of the DNA repair protein Werner helicase (WRN) to E1-24 E2 replicating DNA. CRISPR/Cas9 editing demonstrates that WRN, like SIRT1, regulates the quantity and 25 fidelity of E1-E2 replication. This is the first report of WRN regulation of E1-E2 DNA replication, or a role 26 for WRN in the HPV life cycle. In the absence of SIRT1 there is an increased acetylation and stability of 27 WRN, but a reduced ability to interact with E1-E2 replicating DNA. We present a model in which E1-E2 28 replication turns on the DDR stimulating SIRT1 deacetylation of WRN. This deacetylation promotes WRN 29 interaction with E1-E2 replicating DNA to control the quantity and fidelity of replication. As well as 30 offering a crucial insight into HPV replication control, this system offers a unique model for investigating 31 the link between SIRT1 and WRN in controlling replication in mammalian cells. 32 Importance 33HPV16 is the major viral human carcinogen, responsible for between 3 and 4 % of all cancers worldwide. 34Following infection this virus activates the DNA damage response (DDR) to promote its life cycle, and 35 recruits DDR proteins to its replicating DNA in order to facilitate homologous recombination during 36replication. This promotes the production of viable viral progeny. Our understanding of how HPV16 37 replication interacts with the DDR remains incomplete. Here we demonstrate that the cellular deacetylase 38 SIRT1, which is a part of the E1-E2 replication complex, regulates recruitment of the DNA repair protein 39 WRN to the replicating DNA. We demonstrate that WRN regulates the level and fidelity of E1-E2 40 3 replication. Overall the results suggest a mechanism where SIRT1 deacetylation of WRN promotes its 41 interaction with E1-E2 replicating DNA to control the levels and fidelity of that replication. 42 43 44 HPV16 E1-E2 DNA replication uses translesion synthesis to by-pass replication polymerases on UV 126 damaged DNA (66), and that replication in the presence of DNA damaging agents is mutagenic (48). We 127 now demonstrate that deletion of SIRT1 from C33a cells resulted in an elevation in mutation frequency of 128 3 to 4 fold (Fig 1c). Restoration of SIRT1 expression during E1-E2 DNA replication in the SIRT1 CRISPR 129 knock out cells ...

Werner Syndrome Protein (WRN) Regulates Cell Proliferation and the Human Papillomavirus 16 Life Cycle during Epithelial Differentiation

“…HPV16 is responsible for around 50% of HPV-positive cervical cancers and 80 to 90% of HPV-positive oropharyngeal cancers. We have identified several proteins as being involved in E1-E2 DNA replication, including TopBP1, SIRT1, and WRN, all proteins that have key roles in the DNA damage response, HR, and DNA replication ( 25 – 30 ). Our recent work on WRN demonstrated that deacetylation of this protein by SIRT1 (a protein that also deacetylates TopBP1) promotes binding of WRN to HPV16 origin containing plasmid DNA being replicated by E1-E2 ( 25 ).…”

Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

“…An advantage of the CRISPR approach is that wild-type and mutant plasmids can complement the lesion in transient assays. For example, co-transfection of wild-type WRN plasmid in the replication assay in WRN CRISPR edited cells restores wild-type replication phenotypes [ 46 ]. Therefore, enzymatic and structural functions of host factors controlling replication and repair (WRN, for example) can quickly and easily be determined.…”

“…The second mechanism that resolves the chicken foot structure and promotes replication restart is homologous recombination (HR), a process that may occur without a DNA DSB from single-stranded DNA (ssDNA) generated at the reversed forks [ 38 ]. HPV replication uses HR, and a host of HR factors interact with HPV genomes [ 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ]. The paused forks trigger the DDR via increased stretches of ssDNA that activate ATR and the recruitment of the MRE11-RAD50-NBS1 (MRN) complex that mediates HR and activates ATM; HPV-replicating DNA recruits MRN components [ 24 , 40 , 41 , 42 , 48 , 49 , 50 ].…”

“…First, replication levels increased, at least in part due to increased acetylation and stability of E2. Second, the fidelity of replication was reduced in the absence of SIRT1 (as measured using our blue/white assay) [ 46 , 47 ]. To investigate the reason for the reduced fidelity of replication in the absence of SIRT1, we investigated changes in the recruitment of SIRT1 DNA replication and repair substrates to E1–E2-replicating DNA.…”

“…In the absence of SIRT1, there is an increase in WRN acetylation [ 74 , 75 ]. In the absence of SIRT1 we observed an elevated level of WRN in C33a cells that was hyperacetylated; the acetylation of lysines may prevent the ubiquitination and proteasomal targeting of these residues [ 46 ]. However, the acetylated WRN could not interact with the E1–E2 replicating DNA, perhaps due to the increased negative charge following acetylation.…”