Sae2 and Rif2 regulate MRX endonuclease activity at DNA double-strand breaks in opposite manners

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Protein-Protein Interactions

Background:

• The Mre11-Rad50-Xrs2 (MRX) complex is crucial for detecting and processing DNA double-strand breaks (DSBs).
• MRX activity is regulated by ATP-bound and nucleolytically active post-hydrolysis states.
• Sae2 protein stimulates Mre11 endonuclease activity, and its absence leads to prolonged MRX at DSBs.

Purpose of the Study:

• To investigate the role of Rif2 protein in regulating MRX complex activity at DSBs.
• To elucidate the mechanism by which Rif2 influences Mre11 endonuclease function.
• To understand the interplay between Sae2 and Rif2 in MRX complex regulation.

Main Methods:

• Biochemical assays to assess Mre11 endonuclease activity.
• Analysis of protein-protein interactions between Rad50 and Rif2.
• Site-directed mutagenesis to identify key residues in Rad50-Rif2 interaction.

Main Results:

• Rif2 protein inhibits Mre11 endonuclease activity.
• Rif2 is responsible for the increased retention of the MRX complex at DSBs in Sae2-deficient cells.
• A specific Rad50 residue is identified as critical for Rad50-Rif2 interaction and Rif2's inhibitory effect.

Conclusions:

• Rif2 antagonizes Sae2's function by stabilizing an endonuclease-inactive MRX conformation.
• Sae2 promotes DNA cleavage by stabilizing a post-hydrolysis MRX conformation.
• Rif2 and Sae2 have opposing roles in regulating Mre11 endonuclease activity during DNA double-strand break repair.