Fission yeast Rad52 phosphorylation restrains error prone recombination pathways

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Cellular Stress Response

Background:

• Rad52 is a crucial protein in homologous recombination (HR), facilitating DNA double-strand break repair and replication fork recovery.
• Rad52's functions include promoting Rad51 loading for strand invasion and participating in Rad51-independent pathways like single-strand annealing (SSA).
• Previous studies showed fission yeast Rad52 is phosphorylated by Sty1 under oxidative stress and in Rad51-deficient cells.

Purpose of the Study:

• To investigate the regulation of Rad52 phosphorylation in response to impaired homologous recombination (HR) and its dependence on specific residues.
• To elucidate the role of serine 365 (S365) in Rad52 phosphorylation and its impact on DNA repair and recombination.

Main Methods:

• Analysis of Rad52 phosphorylation in fission yeast mutants lacking key HR proteins (Rad51, Mus81).
• Site-directed mutagenesis of Rad52, specifically mutating serine 365 to glycine (S365G).
• Assessment of spontaneous homologous recombination (HR) rates and sensitivity to hydroxyurea in wild-type and mutant strains.

Main Results:

• Rad52 is constitutively phosphorylated in mus81 null cells, with partial dependence on Sty1.
• Mutation of S365 in Rad52 abrogates constitutive phosphorylation in rad51 null cells and partially reduces it in mus81 null cells.
• The S365G mutation prevents stress-induced phosphorylation of Rad52, indicating distinct phosphorylation sites for different stress conditions.
• The rad52-S365G mutant exhibits increased spontaneous deletion-type recombinants, dependent on Mus81, and heightened sensitivity to hydroxyurea.

Conclusions:

• Different cellular conditions (oxidative stress, HR impairment) trigger phosphorylation of distinct Rad52 residues, mediated by Sty1.
• Serine 365 is a key site for constitutive Rad52 phosphorylation in the absence of Rad51 and plays a role in Mus81-dependent DNA repair.
• Modulation of Rad52 phosphorylation at S365 affects DNA repair fidelity and recombination outcomes, highlighting its regulatory importance.