Figure 2.

The effects of EndoMS-sliding clamp interactions in vitro and in vivo. (A) Alignment of the C-terminal region of EndoMS/NucS proteins. (B) Immunoprecipitation analysis of EndoMS. Cell-free extracts (Input) of the indicated strains were subjected to immunoprecipitation (IP) with anti-EndoMS_Cg antibody followed by immunoprobing with anti-EndoMS_Cg (upper panel) and anti-DnaN_Cg (lower panel) antibodies. (C) The effect of deleting the sliding clamp binding motif in EndoMS_Cg on the mutation rate. The rate of spontaneous rifampicin resistance mutations was determined by fluctuation assay. Error bars show 95% confidence intervals (n = 20). (D) Enhancement of EndoMS_Cg activity with the addition of DnaN_Cg. An in vitro cleavage assay using intact EndoMS_Cg (WT) (lane 2–6 and 13–17) and the EndoMS_Cg-Cdel mutant (lane 7–11 and 18–22) was carried out in the absence (lane 2–11) or presence (lane 13–22) of 1 μM DnaN_Cg. The dsDNA with (+) or without (−) G/T mismatch flanking to A residues (100-bp, 50 nM) were used as substrates. The EndoMS_Cg protein concentration in the reaction mixture is indicated. Substrates and cleavage products were indicated by ‘S’ and ‘P’ on the left of panel, respectively. The percentage of cleaved substrates was indicated on the bottom of the panel. (E) Titration curve of relative cleavage activity and concentration of EndoMS_Cg. Relative cleavage activity was plotted against the concentration of EndoMS_Cg based on the data shown in Supplementary Figure S7A. Experiments were repeated three times and average with standard deviation was shown. (F) DNA substrates containing the indicated mismatch flanking to A residues (100-bp, 50 nM) were incubated with 3 μM EndoMS_Cg and 1 μM DnaN_Cg.