Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

Area of Science:

• Molecular Biology
• Genetics
• DNA Repair

Background:

• DNA double-strand breaks (DSBs) trigger homologous recombination (HR) for repair.
• Processing of DSBs involves 5'-3' nucleolytic degradation to create single-stranded DNA (ssDNA) substrates for Rad51.
• Key factors implicated in DSB processing include the Mre11 complex, Sae2, and Exo1.

Purpose of the Study:

• To elucidate the roles of yeast Exo1 nuclease and Sgs1 helicase in DSB processing.
• To investigate the interplay between Sae2, Exo1, and Sgs1 in generating substrates for homologous recombination.
• To define the mechanistic steps involved in eukaryotic DSB end resection.

Main Methods:

• Genetic analysis of yeast mutants deficient in DSB processing factors (Exo1, Sgs1, Sae2).
• Characterization of DNA repair intermediates accumulating in specific mutant backgrounds.
• Assessment of the impact of mutations on the efficiency of homologous recombination.

Main Results:

• Yeast Exo1 and Sgs1 function in parallel pathways for DSB processing.
• Accumulation of partially resected DSB intermediates in exo1 sgs1 double mutants.
• Sae2 is essential for the initial processing step generating these intermediates.
• Absence of Sae2 leads to accumulation of unprocessed DSBs and failure of homology-dependent repair.

Conclusions:

• DSB processing during homologous recombination follows a two-step mechanism.
• Step 1: Mre11 complex and Sae2 generate an early intermediate by removing oligonucleotides.
• Step 2: Exo1 and/or Sgs1 extensively resect this intermediate to produce ssDNA for Rad51 binding and HR initiation.