Persistent infection with high-risk HPVs cause multiple human cancers; however there are no antivirals to treat these diseases. An increased understanding of the virus-host interactions that regulate the viral life cycle may reveal novel approaches for therapeutic development. Upon infection, HPV genomes transiently amplify to 50- 100 episomal copies per cell that are stably maintained. Epithelial differentiation triggers productive replication, resulting in amplification of viral genomes to 100s-1000s of copies/cell. We have shown that the ATM-dependent DNA damage response (DDR) promotes recruitment of homologous recombination (HR) repair factors (e.g. BRCA1, Rad51) to HPV genomes to facilitate productive replication. Despite an increase in cellular double-strand breaks (DSBs) upon differentiation, HPV genomes are preferentially repaired, though the mechanistic basis for this is unknown. We recently demonstrated that the DDR ubiquitin ligase RNF168 is specifically required for viral genome amplification upon differentiation. RNF168 is recruited to DSBs by ATM signaling. RNF168 plays a critical role in the DDR by catalyzing histone ubiquitination to recruit DNA repair proteins, including 53BP1 and BRCA1, both of which localize to HPV replication foci. How HPV uses RNF168 activity to drive viral replication is unclear, although several recent studies have implicated RNF168 in HR repair. The HPV E7 protein interacts directly with RNF168, disrupting repair at cellular DSBs. E7 may sequester RNF168 from cellular DSBs to direct RNF168 activity to viral chromatin, promoting preferential recruitment of HR factors to viral DNA upon differentiation. However, RNF168-mediated 53BP1 recruitment to DSBs poses a block to HR initiation that HPV must overcome. BRCA1 recruitment to post-replicative chromatin in S/G2 phases antagonizes this block by redistributing 53BP1. We have found that 53BP1 is redistributed at HPV DNA foci upon differentiation, implicating BRCA1 in promoting HR initiation on viral chromatin. Interestingly, our preliminary studies indicate that DNA-PK, a kinase critical for error-prone non-homologous end joining (NHEJ), may contribute to viral genome amplification by repressing cellular DSB repair pathways that could interfere with HR factor recruitment to viral DNA. We hypothesize HPV reshapes the cellular DNA damage response upon differentiation to support HR on viral chromatin. In this proposal, we will test if RNF168 promotes productive replication through HR factor recruitment to viral chromatin and determine if the E7-RNF168 interaction facilitates this process. We will also determine if the HPV-mediated increase in RNF168 protein stability influences viral replication. We will determine if BRCA1 monitors the state of post-replicative viral DNA to direct repair to HR by removing 53BP1. Additionally, we will define the interplay between HR and NHEJ during productive replication by determining if DNA-PK activity provides a cellular environ...