Mechanisms of Mammalian Double-Strand Break Repair

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 DESCRIPTION (provided by applicant): Genome instability in the form of chromosome rearrangements is a hallmark of cancer cells and a driver of tumorigenesis. Mounting evidence indicates that an error-prone alternative form of double-strand break repair called microhomology-mediated end joining (MMEJ) promotes chromosome rearrangements associated with DNA deletions by utilizing sequence microhomology to recombine broken DNA. MMEJ is distinct from the classical non-homologous end joining (NHEJ) pathway since it functions in a Ku and Ligase IV independent manner and is therefore referred to as alternative end joining (alt-EJ). Although MMEJ appears to be the major form of alt-EJ, the central mechanism of this elusive pathway remains unknown. Genetic studies in C. elegans and Drosophila, however, suggest a central role for the atypical A-family DNA polymerase theta (Pol). In preliminary studies, we demonstrate for the first time that the polymerase domain expressed by human POLQ - herein referred to as Pol-performs MMEJ of DNA containing 3' single-strand DNA (ssDNA) overhangs with two or more base-pairs of homology, including DNA modeled after telomeres. We show that MMEJ is specific to Pol, is facilitated by hydrogen bond formation between opposing overhangs, and is dependent on Pol in vivo. Remarkably, we find that Pol exhibits DNA end joining and microhomology annealing activities separately from its replication function. Yet, the polymerase utilizes the opposing overhang as a template in trans to stabilize the DNA synapse. We further find that Pol preferentially performs MMEJ of DNA containing a 5'-terminal phosphate, which demonstrates a functional similarity to X-family polymerases involved in NHEJ. Additionally, we identify a conserved insertion loop domain in Pol that is essential for MMEJ and higher-order structures of the polymerase which likely facilitate DNA tethering. Lastly, we present data suggesting that Pol exhibits terminal transferase activity, which is thought to contribute to MMEJ. We propose to further characterize the biochemical mechanisms of Poland its involvement in MMEJ by developing the following specific aims: 1. To elucidate the mechanism of MMEJ promoted by Pol; 2. To investigate and characterize terminal transferase activity of Pol; 3. To characterize the activities of full-lenth Pol . In summary, these studies will provide new insight into the activities of the atypical A-family DNA polymerase theta, in particular its role in MMEJ of double-strand breaks, and therefore significantly contribute to the DNA repair research community.