Mechanism and regulation of DNA double-strand break repair

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Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or following treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to profoundly detrimental events, such as chromosome loss, deletions, duplications or translocations. Defects in the repair of DSBs cause genomic instability, manifested as immunological, development or neurological defects, and predisposition to cancer. The toxicity of DSBs is exploited for radiation and chemotherapy, as well as targeted therapies directed against specific DNA repair proteins. Thus understanding the mechanisms of DSB repair is of fundamental importance and has practical application for development of new therapeutics and uncovering pathways to resistance. Typically, cells repair DSBs by either homologous recombination (HR) or non-homologous end joining (NHEJ). HR employs extensive homology and templated DNA synthesis to restore the broken chromosome and is considered to be an error-free process. NHEJ directly ligates DSB ends, a mechanism that is potentially error prone due to small deletions or insertions at the junctions. The choice between these two pathways is governed by the cell cycle, which regulates an early step in HR, namely, 5'-3' resection of DSBs. The overall goal of our research program is to decipher the mechanisms of homology-dependent DSB repair, using the yeast Saccharomyces cerevisiae as a model system. The first part of our program builds on our previous studies showing that the conserved Mre11- Rad50-Xrs2 (MRX) complex initiates 5'-3' resection. Specifically, we will use next-generation sequencing to identify the sites of MRX nicking, determine how chromatin structure influences nick site selection and measure the length of resection tracts in cells undergoing HR repair. In addition to controlling end resection, MRX promotes NHEJ, tethers DSB ends and recruits the Tel1ATM kinase to DSBs to activate the DNA damage checkpoint. We will determine the contribution of these diverse functions to genome integrity using specific alleles of MRX components coupled to assays measuring gross chromosome rearrangements. DSBs that arise by replication fork collapse or by erosion of uncapped telomeres have only one free end and are repaired by strand invasion into a homologous duplex DNA followed by replication to the chromosome end (break-induced replication, BIR). The second part of our research program utilizes physical and genetic assays developed in my laboratory to address the mechanism and fidelity of DNA synthesis by BIR.