sea3Δ mutants are not classical recovery mutants
The delay in colony formation in the sea3∆ mutant BIR assay strain suggested that sea3∆ mutants may have a delay in recovery post-DNA damage. Recovery is typically defined as resumption of mitosis after repair is completed and the checkpoint is turned off; recovery mutants demonstrate sustained activation of the DDR despite repair of the DSB (Vaze et al. 2002). A variety of proteins have been implicated in recovery and most are associated with the DNA damage checkpoint or repair (Guillemain et al. 2007; Leroy et al. 2003; Vaze et al. 2002).
We, therefore, first determined whether the sea3Δ mutant BIR assay strain was able to repair the HO-induced DSB via BIR. Like wild-type, it repaired via BIR a majority of the time, as tracked by the sensitivity of the colonies to canavanine and hygromycin (Figure 3A). Additionally, the sea3Δ mutant repaired the DSB as rapidly as wild-type with repair products appearing in the interval between 8 and 10 hr after DSB induction (Figure 3B). Next we determined whether the mutant had a delay in terminating the DNA damage checkpoint postrepair, which is most frequently monitored by examining the pattern of Rad53 hyperphosphorylation after DSB induction. We found, however, that the sea3Δ mutant did not have prolonged Rad53 hyper-phosphorylation as compared to wild-type (Figure 3C). Taken together, as the sea3Δ mutant was able to repair the DSBs and extinguish the DNA damage checkpoint as proficiently as the wild-type, the delay in colony formation post DSB induction was distinct from a classical recovery defect.
Figure 3  Loss of Sea3 does not delay break-induced replication (BIR) repair or extinction of the DNA damage checkpoint. (A) Percent repair types observed in wild-type and sea3Δ BIR assay strain mutants from (Figure 2B), determined by plating on media containing canavanine or hygromycin. Values represent average of two independent trials. (B) AvaI-digested genomic DNA collected from wild-type and sea3Δ BIR assay strain mutants at designated hours postgalactose induction blotted and probed with the CAN1 gene. (C) Rad53 western blot analysis of whole-cell extracts of an equivalent number of wild-type and sea3Δ mutant cells were prepared via the trichloroacetic acid method at designated hours postgalactose induction. Image is representative of two independent experiments.