Extended Data Fig. 6: Human MutLγ binding to single-stranded DNA.

a, Representative image of EMSAs comparing human MutLγ binding to ssDNA and Holliday junctions. b, Quantification of the EMSAs represented in a. Mean ± s.e.m. for three independent experiments are shown. 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 human MutLγ binding to 80-mer DNAs containing single-strand gaps of varying size. To define the minimal single-strand gap that is efficiently bound by MutLγ, 80-nt linear DNAs containing gaps of varying lengths were tested. d, Quantification of the EMSAs represented in c showing mean ± s.e.m. for three independent experiments. Significant binding was not detected for gaps of 5 and 10 nt, but 20 and 40 nt gaps were bound efficiently. Notably, this minimal binding site is similar to that bound by the canonical ssDNA binding protein RPA. e, Representative images of EMSAs for MutLγ binding to an 80mer DNA containing a 40-nucleotide gap, with or without previous incubation with RPA. f, Quantification of the EMSAs represented in e shows that human MutLγ binds a 40 nt gap with almost identical affinity as a 40-mer single-stranded DNA; and that pre-incubation with RPA blocks MutLγ binding. Error bars show mean ± s.e.m. for three independent experiments. g, Representative images of EMSAs for MutLγ binding to pro-HJs and gapped-HJs that were pre-incubated with RPA. To test whether the branched and ssDNA binding activities are autonomous, gHJ and pHJ substrates were pre-incubated with RPA and then used as binding substrates for MutLγ. RPA-gHJ and RPA–pHJ complexes were still readily bound by MutLγ. h, Quantification of the EMSAs represented in g and in Fig. 3a shows that ssDNA and junction binding activities of MutLγ are largely independent. Error bars show mean ± s.e.m. for three independent experiments.