Fig. 5.

Unrestricted fork rotation and excessive DNA catenation cause DNA damage and extended postreplicative repair of replicated chromatids. Cells arrested in alpha factor were shifted to restrictive conditions (YP Raf Gal; 37 °C + 25 µg/mL DOX) and released into media containing nocodazole. (A) Samples from the time points indicated were analyzed by Western blot for phosphorylation of H2A S129 (Pgk1 was used for a loading control) and FACS for DNA content. (B) Samples were assayed for Rad53 activation using the Rad53 autophosphorylation assay. Control samples of MMS (methyl methanesulfonate)-treated exponential top2-td and rad53Δ top2-td cells are also shown. (C) PCNA ubiquitylation was monitored in postreplicative cells (80 min following release) by Western blotting. Blotting for PCNA in MMS-treated cells containing either His-tagged wild-type PCNA or His-tagged K164R confirmed the specificity of the PCNA antibody for monoubiquitylated PCNA (U1) or polyubiquitylated PCNA (U2). (D) Chromatin immunoprecipitation of H2A S129 phosphorylation at two tRNAs, tI(AAU)N1 and tA(UGC)L, and euchromatic loci in isogenic top2-td, tof1Δ, and tof1Δ top2-td strains 80 min after release. ChIP signal was normalized to amplification of input DNA before calculation of the relative change at each locus compared with wild-type cells. Data shown are the mean of three independent experiments ±1 SD.