FIG 3.

Deficiency in Mre11 nuclease generates synthetic sensitivity to DNA-damaging agents in combination with central domain SAE2 mutations. (A) Yeast strains deficient in Mre11 nuclease activity, mre11-H125N (16) and the mre11-H125N sae2Δ double mutant, were complemented with FLAG-tagged Sae2 alleles expressed from a CEN plasmid under the control of the native SAE2 promoter as indicated and tested for MMS and CPT sensitivity as described in the legend to Fig. 2A. (B) FLAG-tagged Sae2 mutants were expressed from low-copy-number CEN plasmids in sae2Δ yeast cells and isolated by using immunoprecipitation with anti-FLAG antibody. Sae2 protein levels in the immunoprecipitates were determined by Western blotting with anti-Sae2 antibody. (C) FLAG-tagged Sae2 was expressed from a low-copy-number plasmid under the control of the native SAE2 promoter in sae2Δ yeast cells. Fivefold serial dilutions of cells expressing the indicated Sae2 alleles were plated onto nonselective medium (control) or medium containing camptothecin (CPT) (5 μg/ml). (D) Yeast strains with the indicated genotypes (mutant alleles integrated into the endogenous SAE2 locus, in the W303 background) were tested for MMS sensitivity as described above for panel A. (E) Yeast strains of the indicated genotypes (mutant alleles integrated into the endogenous SAE2 locus, in the W303 background) were tested for CPT and MMS sensitivity as described above for panel A. (F) ku70Δ sae2Δ yeast strains were complemented with FLAG-tagged SAE2 alleles expressed from a CEN plasmid under the control of the native SAE2 promoter as indicated and tested for CPT sensitivity as described above for panel A.