Molecular mechanisms of DNA double strand break repair by homologous recombination

Project Summary/Abstract DNA double-strand breaks (DSBs) are extremely toxic lesions that sever chromosomes. Cell survival depends on several intricate pathways that repair DSBs efficiently and faithfully, and defects in these pathways drive various cancers and other serious human diseases. The long-term goal of my laboratory is to define the molecular mechanisms of DSB repair and contribute to improved treatments for diseases associated with defective DSB repair. The initial focus of my laboratory will be homologous recombination (HR), a high-fidelity DSB repair pathway whose loss frequently causes breast and ovarian cancer. HR is initiated by 5′3′ resection of broken DNA ends that produces 3′ single stranded DNA (ssDNA) tails. The RAD51 recombinase binds these ssDNA tails and searches for a homologous double stranded DNA (dsDNA) sequence. The RAD51-ssDNA complex then invades the homologous dsDNA to generate a displacement loop (D-loop). The 3′ invading strand is extended by DNA synthesis, templated by the unbroken homologous sequence, to restore sequence information lost at the DSB. In the predominant synthesis-dependent strand annealing (SDSA) subpathway, the D-loop is disrupted to allow annealing of the newly synthesized DNA to the ssDNA arising from the second end of the DSB. Finally, gaps in the DNA are filled to allow ligation, which completes repair. Mechanistic interrogation of HR has remained challenging, as minimal reconstitutions using purified proteins currently lack the complexity to recapitulate multi-step recombination reactions, and cell-based approaches lack the resolution to probe transient HR intermediates. To bridge this gap, I have used Xenopus laevis egg extracts to establish a physiologically complex and biochemically tractable system that robustly recapitulates a complete vertebrate HR reaction in a test tube. Over the next five years, my laboratory will use this unique system to study mechanisms of D-loop metabolism and second end annealing. These transient HR intermediates remain poorly characterized due to a lack of methods that can directly visualize and interrogate them. My laboratory will employ complementary proteomics and real-time single-molecule imaging approaches in egg extracts to investigate the molecular mechanisms that regulate these intermediates with high spatial and temporal resolution. This research program will elucidate fundamental mechanisms of HR that suppress cancer and inform efforts to improve cancer treatments.