Fig. 6: FAN1 inhibits MutSβ-MutLγ through structural and catalytic functions.
From: Mechanism of trinucleotide repeat expansion by MutSβ-MutLγ and contraction by FAN1
a Representative nicking assays with pSam_(CAG)4 DNA and the indicated proteins. Averages shown; error bars, s.e.m.; n = 4 independent experiments. Statistical analysis was performed with a two-tailed t test. Nuclease-dead FAN1 ND contains D960A, D981A, R982A substitutions. Nuclease-dead and MLH1-binding defective FAN1 MIP-MIM ND contains Y128A, F129A, L155A, L159A, D906A, D981A, R982A point mutations. FAN1 ND inhibits DNA cleavage by MutSβ-MutLγ. b Left, protein interaction assay. MutLγ (MLH1-MLH3) was immobilized using anti-MLH1 antibody, followed by incubation with MutSβ (MSH2-MSH3) and/or FAN1 variants, as indicated. MLH1-binding defective FAN1 MIP-MIM contains Y128A, F129A, L155A, L159A point mutations. When FAN1 was included (note that MBP-tagged FAN1 was used), the interaction between MutLγ and MutSβ was diminished. There was no effect on MutLγ and MutSβ interaction when MBP-tagged FAN1 MIP-MIM was used. Top, averages shown; error bars, s.e.m.; n = 3 independent experiments. The data were normalized to the interaction between MutLγ and MutSβ without FAN1. Statistical analysis was performed with a two-tailed t test. ns, non-significant. Bottom, representative Western blot analysis. The cartoon on the right depicts FAN1 disrupting the physical interaction between MutLγ and MutSβ. c, d A representative of three independent nuclease assays with pSam_(CAG)4 DNA and the indicated proteins. The reaction products were analyzed by Southern blotting with probes complementary to the strand opposite the loop, P4 (c) or to the looped strand, P1 (d). FAN1 MIP-MIM, MLH1-binding defective FAN1. FAN1 MIP-MIM ND, nuclease-dead and MLH1-binding defective FAN1. FAN1 ND, nuclease-dead FAN1. On the strand opposite the loop (c), the major DNA incision points by MutLγ are indicated with the orange triangles and an incision point by FAN1 is indicated by the red triangle. DNA incision by MutLγ is inhibited by FAN1 ND (lane 5). The looped strand (d) is cleaved efficiently by FAN1 (lanes 7–9), and its activity is not inhibited by MutLγ. *, DNA not cleaved by ScaI. e pSam_(CAG)4 DNA was reacted with the indicated proteins in the presence of dNTPs, and the reaction products were analyzed by Sanger sequencing and TIDER analysis. The p-value associated with the trace signal is computed using a two-tailed t test, with the standard errors derived from the variance-covariance matrix. Source data are provided as a Source Data file.
