Synthetic viability genomic screening defines Sae2 function in DNA repair

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA double-strand breaks (DSBs) are repaired via homologous recombination (HR), requiring 5' DNA-end resection to generate 3' single-stranded DNA (ssDNA).
• In meiosis, Sae2 and Mre11 facilitate DSB resection by removing Spo11. Their mitotic roles and the cause of DNA damage hypersensitivity in their absence are unclear.
• Cells lacking Sae2 or Mre11 nuclease activity show modest resection defects but significant DNA damage sensitivity, indicating critical but poorly understood mitotic functions.

Purpose of the Study:

• To elucidate the enigmatic mitotic roles of Sae2 and Mre11 nuclease activity in DNA double-strand break repair.
• To identify specific Mre11 mutations that suppress the DNA damage sensitivity of sae2∆ cells.
• To determine the precise mechanism by which Sae2 and Mre11 contribute to homologous recombination in mitosis.

Main Methods:

• Utilized classic genetic suppressor screening combined with high-throughput DNA sequencing to identify suppressive Mre11 mutations.
• Conducted cellular, biochemical, and structural analyses to assess the impact of these Mre11 mutations.
• Investigated the role of Sae2 and Mre11 in DNA-end resection and homologous recombination processes.

Main Results:

• Identified specific Mre11 mutations that significantly suppress the DNA damage sensitivity observed in sae2∆ cells.
• Demonstrated that Sae2 and Mre11 nuclease activity are essential for removing Mre11 from ssDNA at DSB ends during mitosis.
• Showed that Mre11 remaining bound to processed DSB ends impedes strand invasion and homologous recombination.

Conclusions:

• Propose a crucial mitotic role for Sae2 and Mre11 nuclease activity in facilitating Mre11 removal from ssDNA at DSB sites, beyond promoting resection.
• Conclude that the persistence of Mre11 on DSB ends in the absence of Sae2 or Mre11 nuclease activity impairs key steps of homologous recombination, specifically strand invasion.
• Highlight the importance of regulated Mre11 dissociation for efficient mitotic DSB repair and genome stability.