Polymerase Delta Is Required For The Removal Of Short Non-Homologous Dna Flaps During Single-Strand Annealing Repair

Single-strand annealing (SSA) is an error-prone DNA repair pathway that mends double-strand breaks (DSBs) arising between homologous DNA repeat sequences. Repair by SSA requires the removal of 3ʹ-nonhomologous single-strand DNA “flaps”, which can be cleaved by the Rad1-Rad10 endonuclease complex. In the budding yeast, Saccharomyces cerevisiae, Rad1-Rad10 has been proposed to be recruited to SSA repair intermediates by Saw1. However, in vitro Saw1 showed variable affinity for 3ʹ-flaps shorter than 30 nucleotides, and hence the requirement of Saw1 in recruiting Rad1-Rad10 may be flap length dependent. In this work, we engineered yeast strains containing a DNA cassette consisting of an inducible DSB site flanked on both sides by homologous gene repeat sequences. Further, these yeast strains possess fluorescently-labeled proteins that allow us to monitor the localization of DNA repair proteins to the DSB site in our cassette. Strains containing either a wild-type SAW1 gene or deletion of SAW1 were analyzed by fluorescence microscopy to monitor Rad1-Rad10 recruitment during SSA repair. Our results showed that SAW1 is not required for recruiting Rad1-Rad10 to short 3ʹ-flaps (~10 nucleotides). We then corroborated our results using qPCR to quantify the amount of SSA repair products formed after DSB induction, and observed no significant difference between the wild-type and SAW1 deletion mutant at any timepoint tested. We then examined if the 3ʹ→5ʹ exonuclease activity of DNA polymerases (Pol) might be necessary for SSA repair of intermediates bearing short 3ʹ-flaps. For this, we generated yeast strains containing our DSB cassette and expressing exonuclease-deficient alleles of either Pol δ or Pol ε. Using qPCR, we showed that the 3ʹ→5ʹ exonuclease activity of Pol δ is required for the completion of SSA in vivo when repair intermediates contain short 3ʹ-flaps. There was no significant difference at any timepoint tested in SSA product formation when comparing the wild-type and exonuclease-deficient Pol ε yeast strains. Together, our results demonstrate that the 3ʹ→5ʹ exonuclease-activity of Pol δ is chiefly required for processing SSA repair intermediates bearing short 3′-flaps.