Role of spontaneous DNA damage and single‐stranded DNA in generation of enlarged G₂ phase cells in rad52 mutants of Saccharomyces cerevisiae

Abstract

The DNA of living creatures is continuously being damaged by endogenous and exogenous sources and different DNA repair pathways exist to mend the different types of damage. Double‐stranded breaks (DSBs) in the DNA of eukaryotes can be repaired by either homologous recombination (HR) or nonhomologous end‐joining (NHEJ). The initial steps of HR in the budding yeast S. cerevisiae lead to formation of long ssDNA tails at the broken ends. These tails arise through short resection by the Mrx and Sae2 proteins, followed by longer resection by Exo1 and Sgs1‐Dna2 and possibly other enzymes. The tails are subsequently coated with the single‐stranded DNA binding protein complex Rpa, which then recruits other proteins to the site such as DNA damage checkpoint sensor proteins and RAD52 group strand exchange proteins.After exposure to DNA damaging agents, yeast cell cultures exhibit an increase in G2 phase cells due to activation of the dominant DNA damage cell cycle checkpoint. rad52 haploid and diploid mutants grown in normal liquid cell culture display an increase in large‐budded cells. DAPI staining indicated that over 90% of these cells were in G2 phase. Creation of double mutant strains with RAD52 and any of seven different DNA damage checkpoint genes co‐inactivated resulted in loss of the high G2 cell phenotype, suggesting constitutive activation of DNA damage checkpoints in the mutants. rad52 cells were also found to have other unusual characteristics, including (a) larger average sizes than wildtype cells, especially among large‐budded cells, (b) faster cell sedimentation rates, and (c) strongly enhanced light scattering in aqueous suspensions. The role played by formation of long 3′ ssDNA tails at DSBs during HR has been investigated using rad52 double mutants incapable of forming long tails. DNA damage checkpoint protein and single‐stranded binding protein fusions with GFP are also being employed to analyze differences in foci formation within WT and rad52 cells.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.