Project Grant F30CA298183

DETERMINING THE ROLE OF REPLICATION PROTEIN A IN POLYMERASE THETA-MEDIATED END-JOINING - PROJECT SUMMARY MANY FRONTLINE CANCER TREATMENTS, INCLUDING RADIATION THERAPY AND SEVERAL CHEMOTHERAPEUTICS, WORK BY INDUCING DOUBLE-STRAND BREAKS (DSBS) SINCE CANCER CELLS ARE TYPICALLY MORE VULNERABLE TO DSBS THAN HEALTHY CELLS. THUS, UNDERSTANDING THE COMPLEX PATHWAYS CANCER CELLS USE TO REPAIR DSBS IS CRITICAL TO UNDERSTANDING AND ADDRESSING RESISTANCE TO THESE THERAPEUTIC AGENTS, WHICH ALMOST INVARIABLY ARISES. DEFICIENCIES IN THE HIGH FIDELITY DSB REPAIR PATHWAY HOMOLOGOUS RECOMBINATION PROMOTE TUMORIGENESIS. THE RESULTING CANCERS ARE DEPENDENT ON A MUTAGENIC DSB REPAIR PATHWAY CALLED POLYMERASE THETA-MEDIATED END-JOINING (TMEJ). POLYMERASE THETA (POLTh) IS A MULTIFUNCTIONAL ENZYME CONSISTING OF AN N-TERMINAL HELICASE DOMAIN, DISORDERED LINKER DOMAIN AND C-TERMINAL POLYMERASE DOMAIN. POLTh IS ESSENTIAL FOR TMEJ, BUT THE OTHER FACTORS INVOLVED IN THIS PATHWAY REMAIN POORLY DEFINED. WHILE GENETIC SCREENS HAVE REVEALED SEVERAL NON-ESSENTIAL PROTEINS THAT MAY PLAY A ROLE IN TMEJ, THESE SCREENS ARE INCAPABLE OF IDENTIFYING ESSENTIAL FACTORS THAT INTERACT WITH POLTh TO PROMOTE TMEJ. IN THIS PROPOSAL, I WILL DETERMINE THE ROLE OF THE ESSENTIAL, EUKARYOTIC SINGLE STRAND DNA-BINDING PROTEIN, REPLICATION PROTEIN A (RPA), IN TMEJ. BASED ON PARTIAL KNOCKDOWN IN CELLS, RPA WAS INITIALLY THOUGHT TO INHIBIT TMEJ.1 HOWEVER, WHEN I DEPLETED RPA FROM A CELL-FREE TMEJ ASSAY IN XENOPUS EGG EXTRACTS, END- JOINING WAS ABOLISHED. TMEJ WAS RESCUED WITH RECOMBINANT RPA, DEMONSTRATING THAT RPA IS ESSENTIAL FOR TMEJ. THE DEEP LEARNING TOOL ALPHA-FOLD MULTIMER PREDICTS THAT RPA AND POLTh INTERACT VIA TWO INTERFACES - ONE ON RPA1 AND ONE ON RPA2. WHEN I MUTATED THESE INTERFACES ON RPA, REPLICATION WAS UNAFFECTED BUT TMEJ WAS INHIBITED. MUTATING THE CORRESPONDING RESIDUES ON POLTh ALSO ABOLISHED TMEJ. I HYPOTHESIZE THAT THESE INTERFACES ARE NECESSARY FOR POLTh RECRUITMENT AND ANNEALING OF MICROHOMOLOGOUS REGIONS. IN AIM 1, I WILL TEST THE HYPOTHESIS THAT RPA1 BINDS AND RECRUITS POLTh AND THAT THIS ALLOWS POLTh TO BRIDGE TWO NON-HOMOLOGOUS STRANDS. IN AIM 2, I WILL TEST THE HYPOTHESIS THAT RPA2 PROMOTES TMEJ BY STIMULATING THE HELICASE DOMAIN OF POLTh TO SIMULTANEOUSLY BIND DNA, ANNEAL TWO DISTINCT STRANDS AND DESTABILIZE AND REMOVE RPA. UNDERSTANDING THE ROLE OF RPA IN TMEJ WILL PROVIDE FUNDAMENTAL INSIGHT INTO HOW POLTh INTERACTS WITH OTHER PROTEINS TO REPAIR DSBS BOTH IN HEALTH AND DISEASE.