FIGURE 9.

CAF-1-, ASF1A-H3-H4-, PCNA-, and RFC-dependent histone (H3-H4)₂ tetramer deposition suppresses unnecessary degradation of the discontinuous strand that occurs in the reconstituted Pol ϵ-dependent MMR reaction. The data were obtained using the MMR assay. The reaction mixtures contained the indicated proteins and 3′-nicked G-T DNA substrate (0.6 nm). When ASF1A-H3-H4, CAF-1, EXO1, MutLα, MutSα, PCNA, RFC, and RPA were included in the reaction mixtures, their concentrations were 46, 23, 3, 6, 22, 21, 3, and 52 nm, respectively. The Pol ϵ concentration was 1, 2, 5, or 10 nm as indicated. The reactions were carried out at 37 °C for 20 min. A, effects of ASF1A-H3-H4, CAF-1, and the different concentrations of Pol ϵ on MMR. The nine-protein MMR system contained ASF1A-H3-H4, CAF-1, EXO1, MutLα, MutSα, PCNA, RFC, RPA, and Pol ϵ. The data are the averages ± 1 S.D. (n ≥ 3). B and C, representative images that show degradations of the discontinuous strands in the reconstituted Pol ϵ-dependent MMR reactions. DNAs recovered from the indicated reaction mixtures were cleaved with ClaI and then separated on a denaturing agarose gel. After the separation, the ClaI-cleaved DNAs were analyzed by Southern hybridizations with ³²P-labeled probes v2505 (B) and v2531 (C). The data are averages (n ≥ 3).