Saw1 localizes to repair sites but is not required for recruitment of Rad10 to repair intermediates bearing short non-homologous 3′ flaps during single-strand annealing in S. cerevisiae
SAW1 is required for efficient removal by the Rad1–Rad10 nuclease of 3′ non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break (DSB) repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing. Saw1 was shown in vitro to bind flaps with high affinity, but displayed diminished affinity when flaps were short (<30 deoxynucleotides [nt]), consistent with it not being required for short flap cleavage. Accordingly, this study, using in vivo fluorescence microscopy showed that SAW1 was not required for recruitment of Rad10-YFP to DNA DSBs during their repair by SSA when the flaps were ~10 nt. In contrast, recruitment of Rad10-YFP to DSBs when flaps were ~500 nt did require SAW1 in G1 phase of cell cycle. Interestingly, we observed a substantial increase in colocalization of Saw1-CFP and Rad10-YFP at DSBs when short flaps were formed during repair, especially in G1, indicating significant recruitment of Saw1 despite there being no requirement for Saw1 to recruit Rad10. Saw1-CFP was seldom observed at DSBs without Rad10-YFP. Together, these results support a model in which Saw1 and Rad1–Rad10 are recruited as a complex to short and long flaps in all phases of cell cycle, but that Saw1 is only required for recruitment of Rad1–Rad10 to DSBs when long flaps are formed in G1.
KeywordsSaw1 Rad10 Double-strand break repair Single-strand annealing
We thank Drs. Adam Bailis and Michael Lisby for critical reading of the manuscript and Jeff Wagner and Matt Labron from UCLA for gamma irradiator access. This work was supported by NIH grants SC3GM093858 (PLF) and S06GM48680 (EPK) and a CSUPERB Presidents’ Commission Scholarship (MM).
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