Mechanism of DNA double-strand break repair and its regulation

The repair of DNA breaks involves either largely accurate and template-dependent homologous recombination (HR), or template-independent and hence mutagenic non-homologous end-joining (NHEJ). HR is initiated by DNA end resection, which involves controlled degradation of the 5'-terminated DNA strands at DNA breaks. Resection occurs in two distinct steps, engaging short- and long-range nuclease complexes. Resected DNA is not a substrate for NHEJ; hence, processes that control resection help determine the DSB repair pathway choice, and have pronounced impacts on genome stability. The 3'-terminated DNA overhang resulting from DNA end resection subsequently invades a homologous DNA template to prime DNA synthesis that recovers any missing genetic information at the break site. Here, I propose to define elements of HR to understand how cells repair broken DNA and how the process is regulated.

Aim I: Role of RPA and Sae2/CtIP in short-range DNA end resection. The single-stranded DNA-binding protein, Replication Protein A (RPA), is known to play crucial roles to activate long-range DNA resection pathways. However, its function in short-range resection remains undefined. We have obtained preliminary evidence that RPA strongly promotes the exonuclease activity of the yeast Mre11 short-range resection complex. We aim to define the role of RPA, as well as the Mre11 co-factor Sae2/CtIP, in both yeast and human short-range resection through biochemical and cellular experiments.

Aim II: Understanding DSB repair pathway choice. We and others have reported that the Mre11 complex removes the NHEJ factor Ku from DNA ends, effectively channeling non-productive NHEJ intermediates into resection and HR, showing that the DSB repair pathway choice is more flexible than previously thought. Supported by preliminary data, we hypothesize that the Mre11 complex can act also on subsequent NHEJ intermediates but only before DNA synapsis occurs. We aim to explain mechanisms underlying the flexibility in DSB repair pathway choice.

Aim III: Role of PIF1, MCM8-9 and other helicases in HR-linked DNA synthesis. In yeast, Pif1 promotes displacement DNA synthesis by Pold, supporting an HR subpathway termed break-induced replication (BIR). The role of human PIF1 is less understood, despite the importance of BIR for the survival of many cancer cells. We aim to define the function of PIF1 and other DNA helicases such as the MCM8-9 complex during HR-based DNA synthesis.

Aim IV: Mechanisms of heteroduplex rejection. Recombination in vegetative cells primarily occurs between identical sequences on sister chromatids. However, recombination between not perfectly matched (homeologous) sequences also occurs, albeit at lower rates, which may have negative consequences such as a loss of heterozygosity and DNA translocations. The efficacy of homeologous recombination is controlled by DNA helicases and postreplicative mismatch repair proteins, but the relationships between these factors, and mechanisms leading to the disruption of HR intermediates are not known. Using biochemical tools, we aim to define processes that limit the efficacy of homeologous recombination.

Our unique biochemical approach will allow us to gain mechanistic insights into DNA breaks repair process that are inaccessible in traditional cellular experiments.