Localization of the site of MutLα-dependent incision of a MeG-T heteroduplex. (A) HCT116BBR nuclear extract was incubated with the indicated 3’ heteroduplexes for 15 min under repair conditions (Materials and Methods) in the absence or presence of 25 nM MutLα. Recovered DNA was digested with NlaIII, reactions products resolved on a 7 M urea polyacrylamide gel and transferred to HybondN+, which was probed with a 5’-32P-end-labeled oligonucleotide complementary to the nicked strand (a136). After stripping, the membrane was reprobed with oligonucleotide s149, complementary to the template strand. (B) Reactions were carried out as in panel (A), except that the substrate corresponding to the first four lanes was a covalently closed circular MeG-T heteroduplex derived from pSYAH1A. The last 6 lanes correspond to a 3’ MeG-T heteroduplex. MutLα-supplemented reactions were done in duplicate. DNA was pre-treated with AGT as indicated.