The study identifies the roles of Sae2, Exo1, and Sgs1 in DNA double-strand break (DSB) processing during homologous recombination. Sae2, Exo1, and Sgs1 function in distinct steps of DSB resection. Sae2 initiates the processing of DSB ends by trimming them to form an early intermediate, which is then rapidly processed by Exo1 or Sgs1 to generate extensive single-stranded DNA (ssDNA) for Rad51-mediated repair. The Mre11 complex and Sae2 are involved in the initial processing step, generating short ssDNA oligonucleotides. Exo1 and Sgs1 then process these intermediates to produce extensive ssDNA for homologous recombination. In the absence of both Exo1 and Sgs1, partially resected intermediates accumulate, which are poor substrates for homologous recombination. Sae2 deficiency leads to the accumulation of unprocessed DSBs and failure of homology-dependent repair. Sgs1 is required for DSB processing in the absence of Exo1. The absence of Sgs1 results in severe resection defects, as evidenced by the accumulation of unprocessed DSB fragments and the failure to generate extensive ssDNA. The study also shows that Sgs1 functions in parallel with Exo1 in processing DSBs. The results suggest a two-step mechanism for DSB processing: first, the Mre11 complex and Sae2 generate an early intermediate, and second, Exo1 and/or Sgs1 process this intermediate to generate extensive ssDNA for Rad51. The findings highlight the importance of these factors in maintaining genomic stability and preventing chromosomal abnormalities. The study also demonstrates that Sgs1, a RecQ helicase homolog, is essential for DSB processing and that its function is conserved across species, including humans, where it is homologous to BLM, a protein associated with Bloom's syndrome. The study provides insights into the molecular mechanisms underlying DSB processing and homologous recombination in eukaryotes.The study identifies the roles of Sae2, Exo1, and Sgs1 in DNA double-strand break (DSB) processing during homologous recombination. Sae2, Exo1, and Sgs1 function in distinct steps of DSB resection. Sae2 initiates the processing of DSB ends by trimming them to form an early intermediate, which is then rapidly processed by Exo1 or Sgs1 to generate extensive single-stranded DNA (ssDNA) for Rad51-mediated repair. The Mre11 complex and Sae2 are involved in the initial processing step, generating short ssDNA oligonucleotides. Exo1 and Sgs1 then process these intermediates to produce extensive ssDNA for homologous recombination. In the absence of both Exo1 and Sgs1, partially resected intermediates accumulate, which are poor substrates for homologous recombination. Sae2 deficiency leads to the accumulation of unprocessed DSBs and failure of homology-dependent repair. Sgs1 is required for DSB processing in the absence of Exo1. The absence of Sgs1 results in severe resection defects, as evidenced by the accumulation of unprocessed DSB fragments and the failure to generate extensive ssDNA. The study also shows that Sgs1 functions in parallel with Exo1 in processing DSBs. The results suggest a two-step mechanism for DSB processing: first, the Mre11 complex and Sae2 generate an early intermediate, and second, Exo1 and/or Sgs1 process this intermediate to generate extensive ssDNA for Rad51. The findings highlight the importance of these factors in maintaining genomic stability and preventing chromosomal abnormalities. The study also demonstrates that Sgs1, a RecQ helicase homolog, is essential for DSB processing and that its function is conserved across species, including humans, where it is homologous to BLM, a protein associated with Bloom's syndrome. The study provides insights into the molecular mechanisms underlying DSB processing and homologous recombination in eukaryotes.