Mechanisms of Mammalian Double-Strand Break Repair

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DNA polymerase q (Polq) is a unique polymerase-helicase fusion protein that promotes the mutagenic DSB repair pathway called microhomology-mediated end-joining (MMEJ), or alternative end-joining. Polq functions on 3’ single-strand DNA (ssDNA) overhangs generated by 5’-3’ exonuclease resection of DSBs, similar to the homologous recombination (HR) machinery. Polq promotes the survival of cancer cells that are deficient in HR due to BRCA1/2 mutations, and confers resistance to chemotherapeutic agents (i.e. ionizing radiation, etoposide). Thus, elucidating the molecular mechanisms of this relatively newly discovered pathway is a priority. How the large (290 kDa) full-length Polq protein performs MMEJ and is organized at the atomic and molecular level remains unclear. Our recently published in vitro studies indicate that attachment of the polymerase and helicase subdomains of Polq is essential for MMEJ. Additional published preliminary studies demonstrate that full-length Polq behaves primarily as monomers. Furthermore, a newly developed GFP reporter cellular assay reveals the existence of Polq-independent MMEJ mechanisms. We plan to fully elucidate the molecular basis of Polq-dependent MMEJ, solve the structure of full-length Polq using cryo-EM, and identify and characterize Polq- independent MMEJ mechanisms by developing the following aims: 1. To elucidate how Polq subdomains contribute to MMEJ; 2. To solve the cryo-EM structure of full-length Polq; 3. To investigate Polq-independent mechanisms of MMEJ. We expect that these studies will identify novel mechanisms of Polq and previously undiscovered MMEJ processes that likely contribute to genome instability and cellular resistance to genotoxic chemotherapy agents.