Fig. 7: Mms4-Mus81 mutant with associated Mms4-P stabilization delays DNA damage response and causes fragility of replication intermediates.

a Time course experiment analyzing Mms4 species within one cell cycle in WT and mms4Δ541-555 mus81Δ121-135. Logarithmically (log) grown cells expressing Mms4 and Mus81 variants from Tc-ADH1-HA promoters were synchronized in G1 phase with α-factor (αF) and then released in YPD medium in the presence of 1 mM Tetracycline (+Tet). After cells reached G2/M, α-factor was again added to the culture. Samples were taken at the indicated timepoints and the presence of HA-tagged Mms4 species and of Cdc5 was analyzed by western blot. Pgk1 served as loading control. The percentage of phosphorylated Mms4 versus total Mms4 was quantified. Cell cycle progression of the cells during the experiment was followed by flow cytometric analysis. 1N and 2N in gray indicate G1 and G2/M phases, respectively. b Kinetics of Rad53 phosphorylation in WT and the mms4Δ541-555 mus81Δ121-135 truncation mutant (see a). Cells were synchronized in G1 phase and then released in YPD medium containing 0.033% MMS. Samples were taken at the indicated timepoints and analyzed by western blot for Rad53 and for Tubulin, which served as loading control. Three independent experiments (n = 3) were used for the quantification of Rad53 phosphorylation; the bars represent the mean values ± SEM. P values were obtained by using a two-tailed unpaired Student’s t test with Welch’s correction, the asterisks indicate *P < 0.05 and **P < 0.01. P value for 30 min = 0.0018, 45 min = 0.0417. NS stands for not significant. c 2D gel profiles of recombination intermediates isolated from cells of the indicated genotype. SGS1-AID and mms4, mus81 alleles are expressed from Tc-ADH1-HA promoters, not indicated. Cells were synchronized in G1 phase with α-factor and then released in medium containing 0.033% MMS. Auxin was added during G1 arrest and after release to induce Sgs1-AID depletion, monitored by western blot. Pgk1 served as loading control. Cell cycle progression of the cells during the experiment was followed by flow cytometric analysis. Replication intermediates were digested with NcoI and visualized using a radioactively labeled probe specific for ARS305 in 2D gel electrophoresis. Signal intensities were quantified, normalized to the monomer spot, and shown relative to the highest value. Red arrows indicate X-shaped replication intermediates. Source data are provided as a Source data file.