FIG. 4.

Kinase activity of Rad53 mutants. (A) Rad53 immune complex kinase assays. yJKD 415 strains with pRS314 DUN1 13xMYC and pRS316 RAD53 3xFLAG alleles were used for these experiments. (Aa) trans-phosphorylation activity of Rad53 with histone substrate. Rad53 was immunoprecipitated with antibody against FLAG from cells treated with or without HU or MMS and incubated on ice with histone H1 in the presence of [γ-³²P]ATP. Gels were exposed to film overnight (top). Histone H1 was visualized by Coomassie blue staining after SDS-5 to 15% PAGE (bottom). (Ab) Autophosphorylation activity of Rad53. (Top) Autoradiogram of Rad53 autophosphorylation activity; (bottom) Western blot of Rad53. (Ac) Rad9, coimmunoprecipitated with Rad53 and phosphorylated in Rad53 immune complexes. (Top) Autoradiogram of Rad9 phosphorylated by Rad53; (bottom) Rad9 Western blot after IP with anti-Rad53 antibodies. Cells were mock treated (lanes C) or treated with HU (lanes H) or MMS (lanes M) prior to analysis. (B) Rad53 ISA. Experiments were performed as described for Fig. 1B with the same strains. Rad53 was detected by Western blotting following ISA. WT, wild type.