Characterization of POLQ's Function in Replication Rescue

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Project Summary Genomic instability is a hallmark of cancer known to generate genetic alterations during cell division. It alone can drive oncogenesis and evolve cancer cells that no longer “play by the rules”. This is accomplished via introduction of key mutations in tumor suppressor and cell cycle checkpoint genes that allow cancer cells to resist many therapeutic interventions. Notably, the standard of care for many cancers is treatment with DNA damaging agents – chemotherapeutics and radiation therapy – with the goal of damaging cancer cells beyond repair. Thus, understanding the mechanisms by which DNA damage is repaired under these conditions is of the upmost importance for both cancer prevention and its treatment. Amongst DNA damage, the most severe threat to the genome are DNA double strand breaks (DSBs). DSBs have 3 major repair mechanisms which can be deployed in response to their accumulation – homologous recombination (HR), classical non-homologous end- joining, and Theta-mediated end-joining (TMEJ). The latter is upregulated in many cancers, specifically those in which HR genes are mutated (BRCA1/2, PALB2, etc.). However, little is known about the role this pathway plays in repairing breaks which occur during DNA replication, a cell cycle stage in which many cancer therapeutics induce DNA damage. I propose to study the precise role the central protein in TMEJ, POLQ, plays during DNA replication and the genomic consequences of such a role. In my preliminary work, I have established that POLQ deficiency renders cells sensitive to both acute and prolonged Camptothecin (CPT) treatment, a Topoisomerase I poison which induces replication fork collapse. Further, POLQ-null cells display high levels of terminally stalled replication forks after CPT exposure, implying POLQ is capable of repairing forks for replication restart. In this proposal, I aim to study how POLQ contributes to replication fork progression, protection, and recovery after the induction of replicative breaks. Further, I have engineered an inducible broken replication fork reporter system, with which I can measure the kinetics and repair signatures of individual replicative DSBs. Using these experimental approaches, I can dissect the molecular requirements and outcomes of POLQ-mediated repair of broken forks. Finally, I seek to elucidate the role POLQ/TMEJ plays during DNA replication in both HR-proficient and deficient cancers which would provide mechanistic insight into cancer treatments and tumor dynamics. Genome duplication is essential for cancer cell division and thus defining how POLQ repairs replicative breaks and facilitates subsequent replication is essential.