Fig. 5. FAN1-MLH1 interaction promotes repair of CAG/CTG DNA slip-outs.
(A) Scheme of the slipped (CTG)30/(CAG)50 substrate. (B) The (CAG)20 slip-out substrate was incubated with extracts from U2OSGFP-FAN1 cells expressing indicated FAN1 variants. Repair efficiency was quantified by densitometric analysis of Southern blots. Values represent the mean of three independent experiments. Error bars represent ±SD. Statistical significance was calculated by ordinary one-way analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons. **P < 0.005, ***P < 0.0005, ****P < 0.00005. (C) HeLa nuclear extract was incubated with FAN1-derived 60-mer peptides (amino acids 118 to 177) containing wt or mutant MIP-MIM, immunoprecipitated using anti-FAN1 antibody, and immunoblotted with the indicated antibodies. (D) Extracts from HeLa cells were supplemented with increasing concentrations of FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B). (E) Extracts of U2OSGFP-FAN1 wt and U2OSGFP-FAN1 MIP*/MIM* cells were supplemented with indicated FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B).