Fig. 1: EXO1 stimulates MutLγ only in the presence of MutSγ.

a A schematic of the nicking assay used to evaluate the endonuclease activity of MutLγ. Supercoiled DNA (scDNA) was incubated with MutLγ and its co-factors generating nicked DNA, which has different mobility in agarose gel electrophoresis (left). Right, polar plots of reads from GLOE-seq performed on scDNA (5.5 kbp). Briefly, after cleavage by the indicated proteins, all resulting 3’-OH ends were mapped by next-generation sequencing. The DNA cleavage sites in the top and bottom strand are in red and black, respectively. b Nicking assays with MutLγ, MutSγ and RFC-PCNA in the presence of increasing magnesium concentrations. The reaction was carried out in the absence (left) or presence (right) of nuclease-dead EXO1 (D173A). The separation was performed on a 1% agarose gel in the presence of GelRed. c Quantification of nicking assays such as in (b); averages shown. n = 3 independent experiments; error bars, SEM. d Nicking assays with indicated proteins either in the presence of 0.3 mM Mn²⁺ and without RFC-PCNA (left), or in the presence of 0.3 mM Mg²⁺ and RFC-PCNA (right). Top, quantification; averages shown, n = 4 (independent experiments with Mn²⁺) and n = 5 (independent experiments with Mg²⁺); error bars, SEM. Bottom, representative gels. e Nicking assays testing the effect of MutSγ and/or EXO1 (D173A) on the stimulation of MutLγ at 5 mM Mg²⁺ in the presence of RFC-PCNA. Nuclease-dead MutLγ (MutLγ-3ND, D1223N, Q1224K, E1229K) was used as a control. Top, quantification; averages shown, n = 6 independent experiments; error bars, SEM. Bottom, a representative gel. Source data are provided as a Source Data file.