# Uncovering the Roles of DNA-pkcs in Regulating R-loops during HPV Replication

> **NIH NIH F32** · NORTHWESTERN UNIVERSITY · 2024 · $73,828

## Abstract

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.

## Key facts

- **NIH application ID:** 10895174
- **Project number:** 1F32AI183634-01
- **Recipient organization:** NORTHWESTERN UNIVERSITY
- **Principal Investigator:** Conor Templeton
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $73,828
- **Award type:** 1
- **Project period:** 2024-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10895174

## Citation

> US National Institutes of Health, RePORTER application 10895174, Uncovering the Roles of DNA-pkcs in Regulating R-loops during HPV Replication (1F32AI183634-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10895174. Licensed CC0.

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