Project 4: Single-molecule studies defining molecular and cellular requirements for Pol θ

PROJECT SUMMARY: PROJECT 4 This project will investigate mammalian DNA polymerase θ (Pol θ), the defining enzyme for repair of DNA double- strand breaks by polymerase theta-mediated end joining (TMEJ). This is Project 4 (“Single-molecule studies of molecular and cellular requirements for Pol θ”), part of a Program Project titled, “Polymerase theta, genome instability, and cancer”. TMEJ is a mutagenic double-stranded break (DSB) repair process vital for cell survival and therapeutic resistance in cancer, particularly those deficient in homologous recombination (HR) repair, such as BRCA-mutated cancers. Despite its significance as a potential cancer therapeutic target, key mechanistic and functional aspects of TMEJ remain poorly understood, hindering the effective application of targeted therapies. This project aims to fill several fundamental gaps in our knowledge of TMEJ, and explore novel hypotheses by employing an array of innovative biochemical, cellular, and single-molecule techniques and assays to define the key steps and molecular mechanisms of TMEJ. First, we will use biochemical reconstitution and single-molecule assays to investigate some of the key interactions and mechanisms of Pol θ that occur during repair junction formation and later processing steps. Second, we will define the role of Pol θ in the repair of single-ended DSB fork lesions induced by Top1i and its crosstalk with the HR pathway. Third, we will determine Pol θ involvement in suppression of endogenous replication fork damage and replication conflicts. In Aim 1 “Mechanism of TMEJ Repair via Biochemically Reconstituted System” we diverge from the current DSB-centric view of TMEJ by investigating its role in repairing fork lesions, by defining the distinct configurations and molecular requirements involved in repair junction formation, while also investigating the mechanism of its exchange with Pol δ and crosstalk with HR proteins. In Aim 2, “Crosstalk of Pol θ and HR in the Repair of Collapsed Replication Forks”, we will test the hypothesis that Pol θ actively participates in repairing Top1i lesions, and possibly distinct subsets of such lesions that might permit a crosstalk with HR leading to TMEJ when HR is deficient upon BRCA loss. In Aim 3, “Deciphering Pol θ's Role in Suppressing Endogenous Replication Damage”, we will explore the roles that Pol θ plays in suppressing endogenous fork damage at genomic regions prone to persistent replication barriers and breakage, particularly those associated with G4 structures, and test whether the accumulation and/or persistence of such unresolved lesions may underlie the synthetic lethality of Pol θ with several factors. The proposed studies are built on our achievements and developments made in the course of the current funding period, and incorporates new observations, reagents and experimental tools that were derived jointly with the other projects and cores. Our combined diverse approaches include molecular biology, biochemistry, structural biology, and biophysics. Substrates, proteins, and experiments will be designed with Projects 1, 2, and 3, and will be constantly monitored with feedback via Core A. Protein purification will be supported by Core B.