Functional studies and homology modeling of Msh2-Msh3 predict that mispair recognition involves DNA bending and strand separation

Abstract

The Msh2-Msh3 heterodimer recognizes various DNA mispairs, including loops of DNA ranging from 1 to 14 nucleotides and some base-base mispairs. Homology modeling of the mispair-binding domain (MBD) of Msh3 using the related Msh6 MBD revealed that mismatch recognition must be different, even though the MBD folds must be similar. Model-based point mutation alleles of Saccharomyces cerevisiae msh3 designed to disrupt mispair recognition fell into two classes. One class caused defects in repair of both small and large insertion/deletion mispairs, whereas the second class caused defects only in the repair of small insertion/deletion mispairs; mutations of the first class also caused defects in the removal of nonhomologous tails present at the ends of double-strand breaks (DSBs) during DSB repair, whereas mutations of the second class did not cause defects in the removal of nonhomologous tails during DSB repair. Thus, recognition of small insertion/deletion mispairs by Msh3 appears to require a greater degree of interactions with the DNA conformations induced by small insertion/deletion mispairs than with those induced by large insertion/deletions that are intrinsically bent and strand separated. Mapping of the two classes of mutations onto the Msh3 MBD model appears to distinguish mispair recognition regions from DNA stabilization regions.

FIG. 1.

Modeling of Msh3 MBD. (a) Alignment of the MutS homolog protein sequences: Msh3 from S. cerevisiae (y), Ustilago maydis (u), and Homo sapiens (h); Msh6 from S. cerevisiae (y) and H. sapiens (h); and MutS from Escherichia coli (E) and Thermus aquaticus (T). Gray boxes, conserved amino acid residues; green and yellow boxes, amino acid residues differentially conserved between Msh3, Msh6, and MutS; asterisks, residues that were mutated in this study. The secondary structures for E. coli MutS (Protein Data Bank accession number 1e3m [17]), T. aquaticus MutS (Protein Data Bank accession number 1fw6 [15]), and human Msh6 (Protein Data Bank accession number 2o8b [39]) are shown below the amino acid sequence. Blue bars, α helices; peach arrows, β sheets. (b) Model of Msh3 MBD on a +T insertion-containing DNA (red and pink) from the Msh2-Msh6 crystal structure (Protein Data Bank accession number 2o8f [39]). The +T insertion is shown in yellow and black. Regions of low confidence (see Materials and Methods) are shown in white. (Inset) Msh2-Msh6 heterodimer on +T insertion-containing DNA; the Msh6 MBD is in dark blue. (c) Model of Msh3 MBD residues on a +T insertion reveals a steric clash of K158, S201, and possibly, Y157 (blue) with the unpaired T (yellow and black). (d) Possible stacking of Y157 with the bases of the non-insertion-containing strand (pink). The molecular images were generated with the PyMOL program (7).

FIG. 2.

Suppression of the msh3Δ phenotype by alternate amino acid substitutions in msh3 mutant alleles in MMR assays. (a) Patches of msh3Δ msh6Δ strains expressing msh3 alleles were replica plated onto plates lacking leucine and threonine for the −1-nucleotide (−1 nt) hom3-10 reversion assay. Patches of msh3Δ msh6Δ strains expressing msh3 alleles and containing a microsatellite plasmid with an in-frame 4-nucleotide repeat sequence upstream of the URA3 gene were replica plated onto plates lacking leucine and tryptophan and containing uracil and 5-fluoroorotic acid, as shown. (b) Mutation rates caused by msh3 mutant alleles in the frameshift repair assay (open bars) and the 4-nucleotide microsatellite assay (closed bars).

FIG. 3.

Differential effect of msh3 mutations on the removal of nonhomologous tails during double-strand-break repair (DSBR). A wild-type strain and derivatives containing the indicated chromosomal msh3 mutations were analyzed for their ability to repair linear plasmids containing a single-base mismatch (black bars), a 30-base nonhomology tail (gray bars), or a 300-base nonhomology tail (white bars) produced by cleavage of plasmid DNAs by HO endonuclease in vivo. Repair is indicated by retention of the plasmids, and defects in repair are indicated by reduced retention of the plasmids. Each experiment was performed 7 to 10 times, and the error bars indicate the standard deviations of the measurements. The msh3-K160D mutation caused defects in the repair of 1-, 2-, and 4-base insertion/deletion mispairs; and the msh3-Y157S, msh3-Y199A, and msh3-R206A mutations caused defects only in the repair of 1-base insertion/deletion mispairs.

FIG. 4.

Differential effects of msh3 mutant alleles in frameshift repair versus microsatellite stability assays. (a and b) Mutations mapped onto the model of the Msh3 MBD placed on a +T-containing DNA from the Msh2-Msh6 crystal structure (white; Protein Data Bank accession number 2o8f [39]). Red residues, positions that, when they are mutated, cause relative defects that are similar in all MMR assays; yellow residues, positions that cause more severe defects in the frameshift reversion assay than the microsatellite stability assay; orange residues, positions that, depending on the specific amino acid substitution, can cause equivalent defects in all MMR assays or greater defects in the frameshift assay than the microsatellite stability assay. (c) Structure of a DNA containing a +T insertion whose bend is induced by Msh2-Msh6 binding (Protein Data Bank accession number 2o8f [39]). Red, T mispair. (d) Structure of an intrinsically bent DNA containing a +5 A insertion (red; Protein Data Bank accession number 1qsk [8]). The molecular images were generated with the PyMOL program (7). (e) Model of the Msh3 MBD binding to intrinsically bent DNA containing large insertions or inducing and stabilizing nonbent DNA containing small DNA insertions. Recognition likely involves a steric wedge inserting between the DNA strands and stabilization of the DNA bend.