First steps in DNA double-strand break repair: underlying mechanisms and regulation

Cells possess two key pathways for DNA double-strand break (DSB) repair. Non-homologous end-joining (NHEJ) is a fast, but mutagenic pathway that does not need a template. Homologous recombination (HR) is instead a template-dependent process, which is usually very accurate. NHEJ is the first choice of repair in the G1 phase of the cell cycle, whereas HR is more common in S/G2, when sister chromatids are available as templates. Pathway misregulation and aberrant DSB repair in general can lead to mutations and translocations, promoting tumorigenesis. The regulatory control of DSB repair is therefore of paramount importance for genome stability maintenance. My laboratory has been focusing on elucidating the mechanisms of the first steps in HR. We have been primarily using biochemistry to understand the underlying molecular mechanisms of the repair process.Recombination is initiated by controlled resection of the 5’-terminated strands at DSBs, leading to 3’-overhangs, coated by the single-strand DNA binding protein replication protein A (RPA). The first steps in resection are catalyzed by MRE11-RAD50-NBS1 (MRN) and CtIP proteins, followed by either DNA2 of EXO1 nucleases. The processes are conserved in evolution and very similar between human and yeast cells. The resected (overhanged) substrates are no longer suitable for NHEJ, and resection is hence a committing step of HR. The first aim of this proposal follows the footsteps of our previous work and aims to understand the mechanism of DNA end resection in eukaryotes, and its regulation. We plan to determine how the resection complex feels DNA ends, how is resection controlled by newly discovered posttranslational modifications and define the interplay of DNA2 with RPA and CtIP.Downstream of resection, RPA that coats the 3’-overhang is replaced by the RAD51 recombinase, which catalyzes the subsequent search for a homologous template. Once template is identified, the RAD51 nucleoprotein filament invades it, creating a displacement loop (D-loop), in which the resected overhangs pair with one of the template DNA strands. The 3’-end then primes recombinational DNA synthesis, which recovers DNA that had been lost at the break sites or removed by resection. The second aim of this proposal will focus on these downstream steps, including potential functional coupling of resection with RAD51 loading, and mechanisms of helicase involvement in DNA synthesis at D-loops, with a focus on the MCM8-9 complex in promoting DNA synthesis during recombination.