Fig. 4
The activity of the gonococcal Vsr endonucleases in the presence of gonococcal MutL protein. Left part of the figure – the activity of V.NgoAXIII; right part of the figure – the activity of V.NgoAXIV endonuclease. a Examples of electrophoresis profiles of the reaction products resolved in a 10% polyacrylamide gel: (black squares) DNA cleavage by gonococcal Vsr endonuclease in the absence of both MutLNgo protein and 1 mM ATP; (black triangles) DNA cleavage by gonococcal Vsr endonuclease in the presence of MutLNgo protein and 1 mM ATP, molar ratio Vsr:MutL 1:2. These and analogous profiles were used to quantify the efficiency of cleavage of substrate DNAs containing a T:G mismatch, catalyzed by Vsr endonucleases. M – marker GeneRuler 50 bp DNA Ladder (Thermo Scientific). Arrows indicate substrate and reaction products obtained after DNA cleavage by a given Vsr endonuclease. P1 and P2 reaction products; S – substrate DNA. b Analysis of the data obtained and fitted to a first-order rate equation using Origin 8.5 software. The presented results were obtained using M.NgoA302P-sub (GTCGGT/ACCGGC) for V.NgoAXIII and M.NgoA1175P-sub (CTGG/CCGG) substrate DNA for V.NgoAXIV. c Histograms presenting first-order rate constants (kst) obtained for endonucleolytic reactions catalyzed by gonococcal Vsr endonucleases in the presence and absence of the gonococcal MutLNgo protein. Asterisks indicate statistically significant differences, which were calculated using the two-tailed heteroscedastic Student’s t-test (p value < 0.05). Reactions were carried out using 0.15 μM substrate DNA, 0.3 μM Vsr endonuclease and 0.6 μM MutLNgo protein. The experiments were performed in triplicate, and representative electrophoresis profiles and graphs of the data fitted to a first-order rate equation are shown