The genome stability network of the Y-family polymerases

Genome instability is a defining feature of cancer, yet the mechanisms that allow cells to tolerate chronic replication stress without catastrophic chromosome breakage remain incompletely characterized in human systems. Translesion synthesis (TLS) represents a conserved branch of the DNA damage tolerance network that permits replication across bulky DNA lesions and post‑replicative gaps, but in mammalian cells, the functional division of labour among Y‑family TLS polymerases and their broader genome maintenance roles are still not fully resolved. In this thesis, I use an isogenic, p53‑deficient RPE‑1 cell library lacking individual Y‑family polymerases (POLH, POLI, POLK, and REV1) to systematically dissect how each enzyme contributes to replication stress responses, genome stability, mutagenesis, and chemoresistance. First, I describe the generation and validation of this TLS knockout panel and show that loss of specific polymerases yields distinct patterns of spontaneous DNA damage, replication stress, and genome instability in the absence and presence of cisplatin, revealing non‑redundant roles for individual Y‑family members in buffering endogenous and exogenous replication stress. Second, I establish a flow cytometry-based PIG-A mutation assay and a low‑coverage whole‑genome sequencing pipeline to quantify how TLS deficiency reshapes mutation rates and mutational signatures following exposure to a panel of genotoxic agents demonstrating that particular polymerase-lesion combinations can both elevate and suppress mutagenesis in a context‑dependent manner. Third, I implement a sub‑genomic DNA damage response CRISPR screen across the TLS knockout library, under untreated and cisplatin‑treated conditions, to map synthetic lethal and chemosensitizing interactions that define the broader genome stability network of Y‑family polymerases, followed by targeted chemical and genetic validation in RPE‑1 and additional model systems. Together, these approaches reveal that Y‑family TLS polymerases form an interconnected yet modular genome maintenance network that differentially safeguards cells from endogenous replication stress, shapes drug‑induced mutational landscapes, and creates polymerase‑specific vulnerabilities that could be exploited to selectively target cancer cells or to enhance the efficacy of DNA‑damaging cancer therapies.