Extended Data Fig. 8: Cleavage of supercoiled plasmid containing a cruciform structure.

a, Endonuclease assays with pUC(AT) and varying mixtures of human MutLγ, MutSγ and EXO1(D173A), with and without ATP (50 nM each protein, 0.5 mM ATP, 5 mM Mn²⁺ and 5 mM Mg²⁺, incubated at 37 °C for 60 min). Mean ± s.e.m. are shown for three experiments. Under these conditions, pUC(AT) is incised 1.3–1.7-fold more efficiently than the corresponding reactions with pUC19 (also see Fig. 3e). Notably, the linear cleavage product seen with the pUC19 substrate (expected position indicated by L*; see Fig. 1e) was not observed. We suggest that this reflects a more physiological nicking reaction because MutSγ and MutLγ can interact with the cruciform structure. Thus, both the efficiency of nicking and the nature of the incision products are altered when a Holliday junction is present. b, The interdependent nature of stimulation by MutSγ and EXO1(D173A). Representative gel image and quantification of ensemble endonuclease reactions with using pUC(AT) as substrate and Mg²⁺ as the sole metal cofactor. Reactions contained 25 nM indicated proteins, 0.5 mM ATP and 5 mM Mg²⁺ and were incubated at 37 °C for 30 min. Mean ± s.e.m. are shown for three experiments. c, d, Negative control ensemble endonuclease assays for pUC(AT) (b) and pUC19 (c) containing nuclease-dead MutLγ(D1223N). Reactions contained 25 nM indicated proteins, 0.5 mM ATP and 5 mM Mg²⁺ or Mn²⁺ and were incubated at 37 °C for 30 min. Average percent nicking ± s.e.m. is shown for three independent assays.