Extended Data Fig. 3: Purification and characterization of human MutSγ.

a, Representative MutSγ purification steps monitored by 10% SDS–PAGE stained with Coomassie blue. b, Representative images of EMSAs analysing MutSγ binding to 80-mer single and double-stranded DNAs. Binding reactions contained 1 nM ³²P-labelled DNA, 100 mM NaCl and 5 mM Mg²⁺ and were incubated on ice for 10 min. Bound and free DNA species were resolved by non-denaturing 5% polyacrylamide gel electrophoresis and processed for imaging. c, Representative images of EMSAs analysing MutSγ binding to pro-HJ and Holliday junction structures. Terminally ³²P-labelled strands are indicated by asterisks. d, Quantification of the EMSAs represented in b and c showing mean ± s.e.m. for three independent experiments. e, SDS–PAGE analysis of purified nuclease-dead human EXO1(D173A) (10% gel stained with Coomassie). f, Endonuclease assays with varying mixtures of MutLγ, MutSγ and EXO1(D173A), with and without ATP (50 nM each protein, 0.5 mM ATP, 5 mM Mn²⁺ and Mg²⁺ incubated at 37 °C for 60 min). Mean ± s.e.m. are shown for three experiments. g, Negative control endonuclease assays with nuclease-dead human MutLγ(D1223N), MutSγ and EXO1(D173A). Average percentage nicking ± s.e.m. are shown for three experiments.