Online

Vanson, Scott

Cellular, Molecular, and Biomedical Science Graduate Program

2022

Ph. D.

DNA damaging events occur every day in every cell of all living organisms andsome may result in double-stranded breaks (DSB). Human DNA polymerase [theta] (Pol [theta]) is a large, 290 kDa DNA repair enzyme and is the only known protein to contain both a polymerase domain and a helicase-like domain (HLD) as one molecule. Pol [theta] is the key mediator of the error-prone DSB repair pathway, Theta-mediated End Joining. This enzyme has been identified as a potential therapeutic target as it may be conferring a survival advantage to subsets of homologous recombination (HR)-deficient cancers, which display elevated expression levels of Pol [theta] and correlate with poor prognoses. Interestingly, the HLD has repeatedly been shown to lack DNA unwinding activity despite its structural similarity to bona fide helicases, and the underlying basis is unknown. Additionally, there is no published structure of the HLD/DNA complex. We identified two structural features of the HLD, a stunted b-hairpin loop and subdomain 5, which we hypothesized may be responsible for its lack of unwinding. We then generated chimeric constructs of the HLD and related, active helicase Hel308 by swapping their b-hairpin loops in addition to a truncated HLD construct lacking subdomain 5. We measured the ability of these mutants to unwind DNA, hydrolyze ATP, and bind ssDNA. Our results indicate that neither feature is solely responsible for the HLD's lack of duplexed DNA unwinding, as we were unable to restore helicase activity to the HLD. However, we show that the stunted b-hairpin is partially responsible due to the observation that chimeric Hel308 containing the shorter HLD hairpin is markedly less efficient at DNA unwinding and ATP hydrolysis than wild type Hel308. A crystal structure of this chimera shows no evidence of perturbed tertiary structure due to the hairpin substitution. In addition, subdomain 5-truncated HLD is more efficient at ATP hydrolysis than wild type indicating that it may be autoinhibitory. We have also collected X-ray diffraction data on HLD crystals grown in the presence of DNA to approximately 3.2Å. Preliminary models in addition to size exclusion chromatography data indicate that the tetrameric HLD may dissociate into constitutive dimers in the presence of DNA. Additionally, we observed a new dimer interface mediated by the winged helix of subdomain 3. Our work described in this dissertation aims to improve the field's structural understanding of Pol [theta]'s HLD. We hope to aid efforts to design novel small-molecule inhibitors and potentially improve outcomes for patients with HR-deficient tumors.