Characterization of a highly conserved binding site of Mlh1 required for exonuclease I-dependent mismatch repair

Abstract

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.

FIG. 1.

Sgs1, Exo1, and Ntg2 interact with yeast and human Mlh1 through their MIP-box. Yeast two-hybrid assays were performed using either pGBT9-Ntg2(1-380), pAS2ΔΔ-Exo1(400-702), or pAS2ΔΔ-Sgs1(785-1447) as baits. The MIP-box of Sgs1, Exo1, and Ntg2 proteins were mutated at critical amino acids in the motif (Ser, Tyr/Phe, and Phe) toward Ala on the bait constructs. Preys were pACT2-yMlh1(1-769) or pACT2-hMLH1(1-756). Transformants were tested for the production of β-galactosidase in an overlay plate assay. (A) Two-hybrid assays with yeast Mlh1; (B) two-hybrid assays with human MLH1. A representative patch is shown. Yeast two-hybrid assays with bait alone or prey alone were realized and gave white patches.

FIG. 2.

Histogram indicating sequence conservation of Exo1 MIP-box among eukaryotic sequences. The height of the amino acid symbols within a stack indicates the relative frequency of each residue. The figure was generated with the logo HMM program (42) with 27 Exo1 sequences taken from various eukaryotic phyla. This histogram illustrates the high conservation of the MIP-box motif in a region of Exo1 proteins with little overall conservation (see also Fig. S1A in the supplemental material for sequence alignments).

FIG. 3.

Interaction between hEXO1 MIP-box mutants and hMLH1 or hMSH2. (A) GST pull-down results between hMLH1 and hEXO1. Lane 1, beads and IVTT hEXO1-WT; lane 2, GST and IVTT hEXO1-WT; lane 3, GST-hMLH1 and IVTT from pcDNA3.1A(−); lane 4, GST-hPMS2 and IVTT hEXO1-WT; lane 5, GST-hMLH1 and IVTT hEXO1-WT; lane 6, GST-hMLH1 and IVTT hEXO1-S504A; lane 7, GST-hMLH1 and IVTT hEXO1-F506A; lane 8, GST-hMLH1 and IVTT hEXO1-F507A; lane 9, GST-hMLH1 and IVTT hEXO1-FF506AA. (B) Band intensities were analyzed with software VisionWorksLS, where the band intensity for each mutant protein was determined relative to the intensity of hEXO1-WT. The relative interactions between GST-hMLH1 and hEXO1 MIP-box mutants shown in panel B were determined from four independent experiments. (C) GST pull-down results between hMLH1 and hMSH2. Lane 1, beads and IVTT hEXO1-WT; lane 2, GST and IVTT hEXO1-WT; lane 3, GST-hMSH2 and IVTT from pcDNA3.1A(−); lane 4, GST-hPMS2 and IVTT hEXO1-WT; lane 5, GST-hMSH2 and IVTT hEXO1-WT; lane 6, GST-hMSH2 and IVTT hEXO1-S504A; lane 7, GST-hMSH2 and IVTT hEXO1-F506A; lane 8, GST-hMSH2 and IVTT hEXO1-F507A; lane 9, GST-hMSH2 and IVTT hEXO1-FF506AA. (D) The band intensities in panel C were analyzed with software VisionWorksLS, where the band intensity for each mutant protein was determined relative to the intensity of hEXO1-WT. The relative interaction between GST-hMSH2 and hEXO1 MIP-box mutants shown in panel 3C was determined from two independent experiments, except for data regarding hEXO1-FF506AA, which is based on three independent experiments.

FIG. 4.

Deletion mapping of yeast Mlh1 protein domains required for binding of Exo1, Ntg2, or Pms1. Yeast two-hybrid assays were performed with either pGBT9-Ntg2(1-380), pAS2ΔΔ-Exo1(400-702), or pAS2ΔΔ-Pms1(661-873) as baits. Preys are pACT2-Mlh1(483-769) or deletion mutants as indicated. The results of two-hybrid assays are reported with “+” for blue colonies and “−” for white colonies after 24 h at 30°C. Expression of the various truncated Mlh1 proteins was monitored using an antibody against HA epitope of the GAL4-AD-fusion proteins. All proteins were similarly expressed (data not shown).

FIG. 5.

Localization of heterodimerization site S1 and Exo1/Ntg2 interaction site S2 in the C-terminal region of Mlh1 from S. cerevisiae. (A) The 28 positions mutated on Mlh1 and tested for interaction with Exo1/Ntg2 are schematically represented over the Mlh1 sequence. Mutations that disrupt or weaken Mlh1 interactions with Exo1/Ntg2 and not with Pms1 are, respectively, in blue or light blue. Mutation R547A that disrupts Mlh1 interaction with Pms1 and not with Exo1/Ntg2 is in magenta. Mutations with no effect on both interactions are in gray. Magenta stars indicate residues proposed to be involved in heterodimerization site S1 (21). Dashed lines represent MutL regions that are not crystallized (14). (B) Surface representation of the Mlh1 model colored according to site-directed mutagenesis results with the same color code as in panel A. (C) Surface representation of the Mlh1 model colored according to the sequence conservation from low (red) to high (blue) conservation. This representation shows two conserved patches that coincide with regions proposed for heterodimerization site S1 (21) and Exo1/Ntg2 interaction site S2 (the present study). (See Fig. S2 in the supplemental material for ribbon representations of the model.)

FIG. 6.

Physical interactions between the C-terminal region of yeast and human Mlh1 and different peptides containing a MIP-box. The thermograms and binding isotherms of the calorimetric titrations of wild-type or mutant Mlh1 by peptides at 303 K are presented. The corresponding thermodynamic parameters are reported in Table 4. (A) Mlh1(WT) and pNtg2; (B) Mlh1(WT) and pSgs1; (C) Mlh1(WT) and pExo1; (D) hMLH1(WT) and pEXO1; (E) Mlh1(WT) with peptide Exo1 mutant on the MIP-box, pExo1 (FF447AA); (F) Mlh1 mutant on the critical position E682 of site S2 and pNtg2.