Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2↠cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR. Synopsis The DNA double-strand break resection nucleases Sae2 and Mre11 cooperate in yeast meiosis, but their interplay in mitotic cells is still less well understood. A synthetic viability screening approach offers new insights into Mre11 structure and function and Sae2 repair roles. Classic genetic suppressor screening combined with high-throughput DNA sequencing and bioinformatics provides a widely applicable approach to identify non-null suppressor mutations. Specific MRE11 mutations suppress DNA damage sensitivities of cells lacking Sae2. Cellular, biochemical and structural effects of these mutations resolve paradoxes related to Sae2 and Mre11 homologous recombination roles. Sae2 and Mre11 nucleases promote homologous recombination by removing toxic Mre11-bound DNA repair intermediates. Identification of Mre11 mutations suppressing the DNA damage sensitivity of Sae2 mutant cells reveals how these resection nucleases cooperate in mitotic yeast cells.