DNA End Resection

The repair of DSBs by HR requires the processing of the ends to yield 3' single-stranded DNA (ssDNA) tails or overhangs, which are substrates for the Rad51 strand exchange protein. The transition from DSB to ssDNA is also required for activation of the ATR-mediated checkpoint response. Insofar as the resected DNA ends are inhibitory to NHEJ, DNA end-resection is the first step that differentiates HR and NHEJ and therefore constitutes a critical control point in repair pathway choice. Consequently, end resection is cell cycle regulated to ensure that it takes place during the S and G2 phases of the cell cycle when the presence of the sister chromatid provides a template for accurate repair by HR.

Recent genetic studies by our laboratory, and others, have elucidated the molecular details of DSB end resection in Saccharomyces cerevisiae and we proposed two-step mechanism that includes nucleases and helicases. Shortly after the DSB is formed, the highly conserved Mre11-Rad50-Xrs2 (MRX) complex is recruited to DNA ends to exert architectural and catalytic functions. The MRX complex provides the Mre11 nuclease which cooperates with Sae2 to catalyze the first step in DSB processing, the removal of a short oligonucleotide from the 5' end. The initial step is essential for meiotic DSB processing, in which DSBs are formed by the topoisomerase Spo11. Upon DNA cleavage, Spo11 remains covalently attached to the 5' ends of the break presenting a block to resection. Removal of Spo11 from meiotic DSB ends involves a Sae2 and MRX-dependent endonucleolytic step that releases Spo11 bound to a short (10-40 nt) oligonucleotide. In addition, the MRX complex is required to recruit Dna2, Exo1 and Sgs1 to the break site. More extensive resection is carried out by the 5'-3' exonuclease, Exo1, or by the combined activities of the Sgs1-Top3-Rmi1 complex (STR) and Dna2. In the absence of MRX (or Sae2), Exo1 and Sgs1 no processing of DNA ends is detected and this results in a complete block to homology-directed repair. We suggest the first step of resection is required to remove end-blocking lesions and also serves to create a substrate that is poorly bound by Ku committing cells to extensive resection and HR.