DNA ligase activities during base excision repair coordination

PROJECT SUMMARY Base excision repair (BER) is a critical mechanism for preventing the mutagenic and lethal consequences of DNA damage. BER is a multi-step pathway that requires a tight coordination between the repair proteins. The downstream steps of BER pathway involves gap filling by DNA polymerase (pol) β and subsequent nick sealing

by DNA ligase (LIGI and LIGIIIα). This step-to-step coordination is orchestrated by non-enzymatic scaffolding protein X-Ray Repair Cross Complementing 1 (XRCC1) that plays a key role in assembling repair proteins. Although the roles of the individual enzymes are largely studied, how the multi-protein BER complex coordinates

while maintaining the repair efficiency remains unclear. Though often considered an accurate process, the BER can contribute to genome instability if normal coordination breaks down. Failure in the BER pathway coordination could result in the formation of strand-break repair intermediates that are more mutagenic or toxic than the initial

DNA lesions. We hypothesize that inaccurate BER pathway coordination during DNA ligase activities within the multi-protein complex(es) at the downstream steps of the repair response results in genomic instability and cytotoxicity. Our parent proposal will provide the first biochemical and structural

characterization of the BER pathway coordination. We will address the main hypothesis with the following projects: In Project 1, using biochemical and biophysical approach, we will define the molecular mechanism by which polβ and DNA ligases (LIGI and LIGIIIα) execute the repair pathway coordination. Our studies will also

elucidate whether a defective scaffolding function of XRCC1 and cancer-associated polβ variants with altered BER functions could be determinants of defective pathway coordination. In Project 2, using X-ray crystallography and cryo-EM, we will elucidate the features of DNA substrate and ligase interaction that dictate

accurate versus mutagenic outcomes during final nick sealing step at atomic resolution and investigate the large BER multi-protein complexes scaffolded by XRCC1 to gain a novel insight into the structural architecture of the repair pathway coordination. This equipment supplement requests the purchase of the Nikon Ti2-E

microscope equipped with the fully automated, 4 color H-TIRF module, as well as the epi-fluorescence module, to visualize BER coordination and to measure DNA ligase activities at the single-molecule level. This purchase will directly support the experiments in Projects 1 and 2 of our parent proposal. The single

molecule approach using fluorescently labeled repair proteins is highly complementary to the ensemble experiments that we are currently. We will define the dynamics of BER pathway coordination and provide a more comprehensive view of the polβ/ligase interplay scaffolded by XRCC1 than either approach alone would give,

thus strengthening the impact of our project. Gaining an understanding of how DNA damage is coordinately repaired within multi-protein complex, and the ramifications of defective pathway coordination will identify novel steps that can be exploited as targets for future rational drug design toward enhancing human health.