FIGURE 6.
The effects of replacement of wild-type MutLα with an endonuclease-deficient MutLα-E705K on the reconstituted excision-dependent MMR reactions. The MMR assay (“Experimental Procedures”) was used to obtain the data. The reaction mixtures contained the indicated proteins and 3′-nicked G-T DNA substrate (0.6 nm). When present in the reaction mixtures, ASF1A-H3-H4, CAF-1, EXO1, MutLα, MutLα-E705K, MutSα, PCNA, Pol δ, RFC, and RPA were at the final concentrations of 46, 23, 3, 6, 6, 22, 21, 10, 3, and 52 nm, respectively. After a 20-min incubation at 37 °C the reactions were stopped, and DNAs present in the reaction mixtures were recovered and analyzed. Analysis of the degradations of the discontinuous strand (A) and MMR (B) is shown in the indicated reactions. To visualize the degradations of the discontinuous strands, the recovered DNAs were cleaved with ClaI, separated on a denaturing agarose gel, and analyzed by a Southern hybridization with the 32P-labeled probe v2505. The data shown in the graphs are the averages ± 1 S.D. (n ≥ 3) and were obtained by quantification of images including those present in A and B.