Fig. 4: Impaired turnover of phosphorylated Mms4 leads to increased chromatin association and abnormal Mus81–Mms4 nuclease activity in the next G1 phase.

a Chromatin binding of Mms4 in G1 phase in WT, esc2Δ, slx5Δ, and cul8Δ cells. Tc-HA-Mms4 cells of the indicated background were synchronized in G1 phase with α-factor and samples were processed to obtain soluble (SOL) and chromatin (CHR) fractions. Both fractions were analyzed by western blot, using an anti-HA antibody. Orc2 served as a chromatin marker and Tubulin as a control for the soluble fraction. The graph represents the quantification of n = 4 independent experiments. Mms4 protein levels of esc2Δ, slx5Δ, and cul8Δ were normalized to Orc2 levels and are shown relative to WT samples. 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 asterisk indicates *P < 0.05. Compared to WT, P value for esc2Δ = 0.012; slx5Δ = 0.041; cul8Δ = 0.016. b Nuclease activity assay of Mms4. Extracts were prepared from Tc-HA-Mms4 WT, esc2Δ, slx5Δ, and cul8Δ cells synchronized in G1 with α-factor or in G2/M with Nocodazole. Mms4 was immunoaffinity purified from the extracts, and the nuclease activity was assessed by the resolution of a ³²P-labeled substrate (3′FL = 3′ flap). The arrow indicates the labeled product resulting from the nucleolytic cleavage of the substrate. As controls, the nuclease assay was either performed with immunoprecipitated extracts from untagged cells (no tag) synchronized in G2/M or the assay was performed in the absence of extract (no IP). Nuclease activity was quantified as the percentage of the cleaved fragment with respect to the total labeled substrate for WT, esc2Δ, slx5Δ, and cul8Δ in G1 and G2/M and then deriving the ratio of activity in G2/M versus G1. Cell synchronization was confirmed by flow cytometric analysis. 1N and 2N in gray indicate G1 and G2/M phases, respectively. nt stands for nucleotide. Source data are provided as a Source data file.