Extended Data Fig. 2: MutLγ endonuclease activity with various metal cofactors and stimulation by ATP.

a, Representative gel image showing MutLγ endonuclease assays with various metal ions (100 nM MutLγ and 5 mM indicated divalent metal ions, incubated at 37 °C for 90 min). Migration positions of supercoiled (S) plasmid and nicked (N) product are shown. For reference, plasmid linearized (L) with BamHI is also shown. Mean ± s.e.m. are shown for three experiments after subtracting background nicked product from no-protein controls. b, Representative endonuclease assays with 100 nM MutLγ in 5 mM Mg²⁺ or Mn²⁺ with the addition of various second metal cofactors (5 mM). Reactions were incubated at 37 °C for 90 min. Metals other than Mg²⁺ compete with Mn²⁺ to inhibit MutLγ endonuclease activity. Mean ± s.e.m. are shown for three experiments. c, Representative gel image and quantification of human MutLγ endonuclease activity with and without ATP and metal cofactors (100 nM MutLγ, 0.5 mM ATP and 5 mM Mn²⁺ and/or Mg²⁺ incubated at 37 °C for 60 min). Mean ± s.e.m. are shown for five experiments. d, MutLγ endonuclease activity with increasing ATP concentration. 100 nM MutLγ was incubated with 5 mM Mn²⁺ and Mg²⁺ and indicated concentrations of ATP at 37 °C for 60 min. Endonuclease stimulation was optimal with 0.5 mM ATP, while higher concentrations were inhibitory. Mean ± s.e.m. are shown for three independent experiments.