Project Summary Human papillomaviruses (HPVs) remain a significant threat to human health as over 5 million people in the US are infected annually, and HPVs account for ~5% of all human cancers. Over 400 subtypes of HPV have been identified, with 12 classified as high-risk strains accounting for all known HPV-caused cancers. The HPV lifecycle is intimately coupled to the cell cycle, DNA damage response (DDR) activation, and differentiation status of the infected cell. Upon infection, HPV induces dsDNA breaks and activates the DDR through members of the phosphatidylinositol 3-kinase-related kinases (PIKK): ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad-3 related (ATR). ATM and ATR activation are essential for the HPV lifecycle; however, a third member of the PIKK family, the DNA-dependent protein kinase catalytic subunit (DNA-pkcs), remains unstudied. My preliminary studies have shown that in cells harboring HPV episomes, DNA-pk is activated to high levels. When DNA-pk activity is ablated using small molecule inhibitors, viral episomal levels are significantly reduced in undifferentiated and differentiated keratinocytes. These data support an important role in DNA-pkcs activation in the HPV lifecycle. Furthermore, the frequency of dsDNA breaks and R-loop levels increase when DNA-pk is inhibited. R-loops are trimeric nucleic acid structures consisting of hybridized RNA:DNA strands and a displaced DNA strand. They form on actively transcribed genes and mediate proper transcription. However, improper formation or resolution of these structures leads to transcription replication conflicts and DNA breaks. I have shown that R-loop levels were elevated in high-risk HPVs and tumors and formed on the HPV genome at high numbers. DNA-pk inhibition caused increased R-loop formation in HPV positive cells, while inhibition of the other PIKKs, ATR and ATM, did not. The central hypothesis of my project is that DNA-pk activation is necessary to maintain R-loop homeostasis within HPV positive cells. First, I will study why DNA-pk activation is important in the HPV lifecycle. I will apply genetic techniques to ablate DNA-pkcs activity and examine how the HPV lifecycle is affected while also assessing how DNA-pk is activated within HPV positive cells. Second, I will analyze how DNA-pk mitigates R-loop formation. Through DRIP-seq and RNA-seq, I will identify where R-loops form when DNA-pk activity is ablated and which R-loop-containing genes have altered transcript levels. R-loop resolving enzymes will be examined for reduced binding to viral and cellular genome regions and whether DNA-pk inhibition alters their steady state levels within HPV positive cells. This project will identify key roles facilitated by DNA-pk in HPV replication while shedding light on important functions of DNA-pk in R-loop functions and cellular biology.