Extended Data Fig. 1: Human MutLγ purification and endonuclease activity.
From: PCNA activates the MutLγ endonuclease to promote meiotic crossing over
a, Human MLH1 and MLH3 expression constructs highlighting domain structure and affinity-purification tags: MLH3 ID, human MLH3 interaction domain; endo, endonuclease domain; MBP, maltose binding protein; PP, PreScission protease cleavage site; 10His, deca-histidine tag. Sequences of the conserved metal binding site for (1) wild-type and (2) the nuclease-dead D1223N mutant of MLH3 are also shown. b, Representative MutLγ purification steps monitored by 10% SDS–PAGE stained with Coomassie blue. Amylose enriched fractions were treated with PreScission Protease, to cleave the maltose-binding protein tag, and then subjected to Ni-NTA affinity purification. c, d, Endonuclease assays for varying concentrations of MutLγ and MutLγ(D1223N) (0–300 nM protein with 5 mM Mn2+ (c) or Mg2+ (d)) incubated at 37 °C for 60 min. e, Quantitation of experiments represented in c and d. Mean ± s.e.m. are shown for three experiments after subtracting background nicked product from no-protein controls.
