The study investigates the mechanisms of DNA double-strand break (DSB) processing in yeast, focusing on the roles of Sae2, Exo1, and Sgs1. DSBs are potentially lethal lesions that can lead to chromosomal abnormalities if not repaired correctly. The Mre11 complex, Sae2, and Exo1 are known to play crucial roles in DSB processing, but the exact mechanisms are not fully understood. The authors demonstrate that Sae2 and Exo1 function in different steps of DSB processing. Sae2 is involved in an early step where it removes a small oligonucleotide from the DNA ends to form a minimally resected intermediate, which is then processed by Exo1 or Sgs1 to generate single-stranded DNA (ssDNA) that serves as a substrate for homologous recombination. In the absence of Sae2, the Mre11 complex, and/or Exo1, unprocessed DSBs accumulate, leading to failure in homology-dependent repair. The study also shows that Sgs1, a RecQ helicase homolog, is essential for DSB processing in the absence of Exo1. The results suggest a two-step mechanism for DSB processing: first, Sae2 and Mre11 complex remove a small oligonucleotide from the DNA ends, and second, Exo1 and/or Sgs1 rapidly process these intermediates to generate extensive ssDNA. These findings provide insights into the general mechanisms of DSB processing in eukaryotes and highlight the importance of these factors in maintaining genome integrity.The study investigates the mechanisms of DNA double-strand break (DSB) processing in yeast, focusing on the roles of Sae2, Exo1, and Sgs1. DSBs are potentially lethal lesions that can lead to chromosomal abnormalities if not repaired correctly. The Mre11 complex, Sae2, and Exo1 are known to play crucial roles in DSB processing, but the exact mechanisms are not fully understood. The authors demonstrate that Sae2 and Exo1 function in different steps of DSB processing. Sae2 is involved in an early step where it removes a small oligonucleotide from the DNA ends to form a minimally resected intermediate, which is then processed by Exo1 or Sgs1 to generate single-stranded DNA (ssDNA) that serves as a substrate for homologous recombination. In the absence of Sae2, the Mre11 complex, and/or Exo1, unprocessed DSBs accumulate, leading to failure in homology-dependent repair. The study also shows that Sgs1, a RecQ helicase homolog, is essential for DSB processing in the absence of Exo1. The results suggest a two-step mechanism for DSB processing: first, Sae2 and Mre11 complex remove a small oligonucleotide from the DNA ends, and second, Exo1 and/or Sgs1 rapidly process these intermediates to generate extensive ssDNA. These findings provide insights into the general mechanisms of DSB processing in eukaryotes and highlight the importance of these factors in maintaining genome integrity.