FIG 5.

(A) Immunoprecipitation of TAP-Brc1 or TAP-Brc1-HYP307-309GFG ectopically expressed from pREP41 plasmid in the wt or Nse6-Myc strain. Mutations in Brc1 BRCT domain 4 weaken its interaction with Nse6. (B) Sensitivity of brc1Δ to MMS is rescued by brc1⁺ and brc1-W248F, P301G but not by a brc1-HYP307-309GFG mutant. All Brc1 variants were expressed from the pREP41 plasmid. The strains were cultured in minimal medium lacking leucine and spotted onto YES medium with or without MMS at 30°C. (C) Effect of Brc1 dosage on Smc5-Smc6 mutant genotoxin sensitivity. Ectopic expression of Brc1 in smc6-74, nse6Δ, nse5Δ, and wild-type strains was analyzed in a spot assay. Overexpression of Brc1 rescued the MMS sensitivity of smc6-74 but not nse5 or nse6 deletion. vector, pREP41; brc1⁺, pREP41-NTAP-Brc1. (D) Spot assay of nse6Δ, brc1Δ, and the double nse6Δ brc1Δ mutant strains. Cells were spotted onto YES medium containing the indicated concentrations of genotoxic agent at 32°C. The double-mutant nse6Δ brc1Δ strain showed higher sensitivity to genotoxic stress than the single-mutant parental strains.