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Wells, James Philip

University of British Columbia

DNA replication is a vulnerable time for genome stability maintenance. Endogenous add oncogenic stressors can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids called R-loops. The capacity for unscheduled R-loops to collapse stalled replication forks and induce genome instability has become apparent. In this work, I provide an overview of the causes and cellular responses to replication stress, detailing the importance of replication checkpoint signaling, replication fork remodelling, and the clinical relevance to cancer development. Recently progress has been made towards identifying R-loop and conflict regulators, however, our understanding of this regulation is incomplete. Building off past findings that utilized a genome-wide interaction screen in budding yeast lacking key R-loop degrading RNase H enzymes, I explored a network of potential R-loop tolerance genes. PCNA is a complex replisome component with over 200 binding partners and was identified as a hit alongside RAD18 and ATAD5 (Elg1 homolog), key factors required for PCNA ubiquitin regulation. Once ubiquitinated, PCNA catalyzes several downstream repair pathways. I validated the roles of these genes in R-loop biology, identifying an accumulation of DNA:RNA hybrids in several human cell lines after gene silencing and in conjugation-incompetent PCNA K164R mutants. In both RAD18- and ATAD5-deficient cells, I observed higher levels of replication stress, DNA damage, and transcription-replication conflicts, dependent on R-loops, transcription, and replication. To gain mechanistic insight into how RAD18 tolerates transcriptional stress, I utilized stressors aphidicolin and pladienolide B to stall replication and disrupt the R-loop landscape respectively. I found that RAD18 localization was dependent on these transcriptional stressors and R-loops. I explored the mechanistic connection between RAD18 and members of the Fanconi anemia (FA) pathway, showing DNA:RNA hybrid epistasis. RAD18 was important for recruitment of FANCD2, the key effector molecule with established roles in resolving R-loop-induced stress, to R-loop prone loci and common fragile sites. These findings expand upon a published connection between RAD18 and the FA pathway by establishing a mechanism involving RAD18 in opposing transcription-associated genome instability through FANCD2 recruitment. The result of my work establishes PCNA ubiquitination as a novel regulatory axis for the tolerance of transcriptional stress.

Text

2023-04-19

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84317

Interdisciplinary Oncology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/