The study identifies Sgs1 helicase and nucleases Dna2 and Exo1 as key factors in the resection of DNA double-strand breaks (DSBs) in eukaryotic cells. The Mre11-Rad50-Xrs2 (MRX) complex initiates 5' strand degradation, while Sgs1 and Dna2 are responsible for long-range 5' strand resection, generating 3' single-stranded DNA tails. Exo1 is required for resection in the absence of Sgs1 or Dna2. The study provides a comprehensive model of the early steps of DSB repair, showing that Sgs1 and Dna2 work redundantly to process 5' strands, with Sgs1 playing a critical role in extending 3' single-stranded DNA. The MRX complex is involved in the initial processing of DSBs but not in the long-range resection. The study also shows that the absence of Sgs1 and Exo1 leads to impaired DNA damage checkpoint activation and reduced gene conversion efficiency. Dna2 is identified as a second nuclease involved in 5' strand resection, working redundantly with Exo1. The study highlights the importance of multiple pathways in DSB repair and the role of different nucleases in processing 5' strands. The findings suggest that the resection pathway is conserved from bacteria to mammals, with similar mechanisms involved in DNA repair. The study provides insights into the complex interplay between different proteins in the repair of DNA damage and the regulation of the DNA damage checkpoint.The study identifies Sgs1 helicase and nucleases Dna2 and Exo1 as key factors in the resection of DNA double-strand breaks (DSBs) in eukaryotic cells. The Mre11-Rad50-Xrs2 (MRX) complex initiates 5' strand degradation, while Sgs1 and Dna2 are responsible for long-range 5' strand resection, generating 3' single-stranded DNA tails. Exo1 is required for resection in the absence of Sgs1 or Dna2. The study provides a comprehensive model of the early steps of DSB repair, showing that Sgs1 and Dna2 work redundantly to process 5' strands, with Sgs1 playing a critical role in extending 3' single-stranded DNA. The MRX complex is involved in the initial processing of DSBs but not in the long-range resection. The study also shows that the absence of Sgs1 and Exo1 leads to impaired DNA damage checkpoint activation and reduced gene conversion efficiency. Dna2 is identified as a second nuclease involved in 5' strand resection, working redundantly with Exo1. The study highlights the importance of multiple pathways in DSB repair and the role of different nucleases in processing 5' strands. The findings suggest that the resection pathway is conserved from bacteria to mammals, with similar mechanisms involved in DNA repair. The study provides insights into the complex interplay between different proteins in the repair of DNA damage and the regulation of the DNA damage checkpoint.