SAW1 is increasingly required to recruit Rad10 as SSA flap-length increases from 20 to 50 bases in single-strand annealing in S. cerevisiae

SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were similar to 10 nt, but it was required when flaps were similar to 500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from similar to 20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about similar to 20 nt and is completely required at similar to 50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the similar to 20-50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.

Automated summary

SAW1 is crucial for the efficient removal of 3' non-homologous DNA ends (flaps) by Rad1-Rad10 nuclease in S. cerevisiae, specifically during single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA) modes of double-strand break repair. In vitro studies have shown increasing affinity of Saw1 for flap DNAs as flap lengths vary, and in vivo investigations using fluorescence microscopy have demonstrated the increasing necessity of SAW1 for Rad10 recruitment as flap length increases. Saw1 is present at repair sites even when not required, showing its important role in SSA repair in the 20-50 nt flap range.