(A) Before sporulation and dissection, TLC1/tlc1::LEU2, NPL3/npl3::KanR diploid cells (yJL325) were transformed with plasmids. Only the tlc1 npl3 strains are shown. Plasmids expressed no Npl3 (CV for control vector), 6xHis-tagged WT or mutant Npl3 (Npl3-F160L has mutated RRM1, Npl3-SNK has mutated RRM2, Npl3-LSNK has mutated RRM1 and RRM2, and Npl3-RK1-15 has KGGs in place of RGGs). Senescence assay was performed as described but in SC-URA medium. Error bars represent standard error of the mean, N = 3 – 5. (B) Cells from survivors were expanded; 6xHis-tagged WT and mutant Npl3 were purified by nickel column. Proteins were analyzed by SDS-PAGE and immunoblotted with anti-6xHis antibody.