Absence of Sea3 slows colony formation after DSB induction
As the sea3∆ mutation slowed the appearance of BIR-dependent survivors in the absence of telomerase, we wanted to determine whether Sea3 specifically impacted BIR. To assess this, we deleted SEA3 in a strain created previously to analyze BIR at an induced DSB (Lydeard et al. 2007). In this strain, CAN1, which confers sensitivity to canavanine and is present on the nonessential telomere-proximal end of the recipient chromosome (Chr V), is truncated by the insertion of an HO endonuclease site flanked by a hygromycin resistance (HPH) reporter (Figure 2A). In addition, a 5′ truncation of CAN1 is inserted on the donor chromosome (Chr XI), resulting in 1157 bp of CAN1 homology between the donor and recipient chromosomes. The HO endonuclease is under the control of a galactose inducible promoter. Upon exposure to galactose, the DSB generated at the HO site on Chr V is repaired via BIR, resulting in a full-length CAN1 gene and loss of the hygromycin reporter. When we deleted SEA3 in this strain, we noted a consistent 2-d delay in the formation of quantifiable colonies on galactose compared with wild-type, whereas the sea3∆ mutants formed colonies comparably with wild-type on glucose (Figure 2B and Figure S2, uninduced). The overall viability on galactose, however, was equivalent to wild-type (Figure 2C). Importantly, the effect on galactose was attributable to loss of the SEA3 gene product as it was complemented by expression of SEA3 on a CEN plasmid (Figure S3A).
Figure 2  Loss of Sea3 impacts colony formation in the break-induced replication (BIR) assay strain and on bleomycin. (A) BIR assay strain (Lydeard et al. 2010). An HO cut site (HO), marked with HPH, is integrated into the CAN1 gene (represented as CA) on chromosome V, deleting the 3′ portion of CAN1. The CAN1 donor (represented as AN1), which shares 1157 bp of homology to the CAN1 gene, is integrated into chromosome XI. Sites marked with A indicate AvaI sites used for monitoring BIR repair in Figure 3B. (B) Platings for single colonies of wild-type and sea3Δ mutants in the BIR assay strain on YPD and YPGal. (C) Percent viability of wild-type and sea3Δ mutants as a ratio of number of colonies on YPGal divided by the dilution factor and then divided by the number of colonies on YPD. Values represent average of two independent trials and error bars indicate standard error of the mean (SEM). (D) Platings for single colonies of wild-type and sea3Δ mutants in the YPH274 genetic background on YPD and YPD + 3.5 μg/mL bleomycin. To determine whether the growth delay was caused by a general sensitivity to galactose (rather than the DSB induced by galactose), we took sea3Δ mutants in the BIR strain background that had been plated previously on galactose, had undergone BIR repair of the DSB and, therefore, would not sustain another HO-induced DSB when replated on galactose, and compared their growth on galactose to sea3Δ mutants that had not been previously exposed to galactose and, therefore, would undergo DSB induction when plated on galactose. If the growth delay of the sea3Δ mutants on galactose were simply due to a general sensitivity to galactose, then the presence or absence of a cleavable HO cut site would not matter and both types of sea3Δ mutants would be equally sensitive to galactose. We found, however, that when the sea3Δ mutants that were originally plated on galactose were re-plated on galactose, quantifiable colonies appeared sooner (Figure S2; see also Figure 5A and Figure S8A), suggesting that the delay in colony formation in the sea3Δ mutant was not simply due to a sensitivity to galactose.
To further explore the growth delay in the absence of Sea3 after DSB induction, we examined the growth of sea3Δ mutants in the YPH274 genetic background on the DSB-inducing agent bleomycin. The sea3∆ mutant strain was sensitive bleomycin and, again, formed colonies more slowly than wild-type (Figure 2D). Growth of the sea3∆ mutant strain on galactose was comparable with the wild-type (Figure S3B), thus, clearly demonstrating a growth defect attributable to DSBs and independent of galactose. Additionally, like the delay in the sea3∆ BIR assay strain background when plated on galactose, the delay in colony formation in the sea3∆ mutant strain on bleomycin was complemented by expression of SEA3 on a CEN plasmid (Figure S3B). Therefore, absence Sea3 resulted in delayed growth upon DNA damage in two different genetic backgrounds—either via an inducible DSB in the BIR assay strain or by the DNA-damaging agent bleomycin in the YPH274 background.