Molecular mechanisms of pathway choice in DNA double strand break repair

Project Summary DNA double strand breaks (DSBs) are a particularly toxic form of DNA damage. Even a single DSB can lead to cell death. Our cells use a number of intricate repair pathways to repair DSBs. For the majority of breaks that occur in our cells a pathway known as non-homologous end joining (NHEJ) is used to effectively “glue” the broken strands back together. Alternatively, a strand at each DNA end is “chewed back” in a process known as resection which generates single-stranded DNA overhangs. Multiple pathways act on these overhangs including homologous recombination (HR) and microhomology mediated end joining (MMEJ). HR is an intricate mechanism which uses a sister chromatid to direct repair in a high-fidelity manner while MMEJ is a mutagenic pathway whose mechanism more closely resembles NHEJ. Proper selection of these repair pathways is critical for human health as misuse is correlated with gross chromosomal changes that can result in cancer. Often cancer cells are deficient in DNA repair pathways which allows them to rapidly acquire traits not normally associated with healthy cells. These repair deficiencies also make them vulnerable to targeted cancer therapies. This proposal seeks to develop a better molecular understanding of how cells choose between these different DSB repair pathways. Experiments in cells have identified a number of proteins that play a role in this molecular decision-making process, but we lack an understanding of how these proteins work together. Traditionally, such knowledge is gained by purifying individual proteins and combining them together to reconstitute a biological process. However, given the sheer number of proteins, it is currently untenable to take such an approach. We have recently validated a cell-free extract made from the eggs of the frog Xenopus laevis as system that recapitulates DSB pathway choice. Combining this physiological biochemical system with powerful imaging approaches to study the dynamics of DNA DSB repair proteins at the single-molecule level, we will elucidate the molecular basis of DSB repair pathway choice. Specifically, we will work to clarify how the NHEJ factor Ku, which antagonizes DNA end resection, is removed from DNA ends (Aim 1); and how the MMEJ polymerase Pol θ competes with long-range resection on partially resected overhangs (Aim 2). Finally, we will elucidate how the multi-functional Pol θ uses its diverse enzymatic activities to synapse DNA ends and search for microhomology (Aim 3). Our studies will reveal significant new insights into DSB repair pathway selection and regulation. These insights may enable novel ways to alter the balance between these repair pathways which could have applications in gene editing or in therapies for cancers deficient in DSB repair.