Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae

Abstract

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.

Figure 1.—

Mismatch repair assays of MSH2 missense alleles. (A) Qualitative functional assays. The phenotypes of the strains expressing the msh2 missense substitutions are compared to the wild type (WT) and msh2 null (Δ) controls to determine the mismatch repair efficiencies in standard canavinine (CAN) (Reenan and Kolodner 1992) and 5-fluororotic acid monohydrate (FOA) resistance plate assays (Henderson and Petes 1992). The missense strains are listed according to the amino acid change; e.g., L521P denotes that at position 521 the leucine codon was changed to a proline codon. Uniform growth on medium lacking histidine and tryptophan (–HIS –TRP) serves as a replica-printing control. (B and C) The frequency of missense mutations resulting in pseudo-wild-type (B) or in mismatch repair-defective (C) phenotypes. The codon number scale on the x-axes is the same for B, C, and D. The scale for frequency corresponding to B and C on the y-axes differ. (D) Conservation among MutS homolog 2 proteins. A CLUSTAL W (version 1.82) multiple sequence alignment of human, mouse, worm, fly, mold, and yeast MSH2 was used in conjunction with the Evolutionary phylogenetic SHADOWing of closely related species (Ovcharenko et al. 2004) to generate a plot showing the degree of conservation (substitutions per amino acids) across the codon positions. The graph is plotted to emphasize the conserved regions of MSH2.

Figure 2.—

Stability alleles defined. (A) Sample immunoblot to detect decreased steady-state levels of Msh2. Protein extracts were fractionated on a 7% separating gel and after blotting, the detection of Msh2 expressing the HA epitope was accomplished as described in the materials and methods. The positive control included the wild-type Msh2-HA protein extract from a msh2Δ strain expressing MSH2 from pMSH2 (WT). The no-epitope control extracts were from a msh2Δ strain harboring the pRS413 vector (Δ). The msh2 alleles were expressed from a plasmid identical to pMSH2 except for the relevant encoded missense mutation. The denoted change is marked above the corresponding lanes of the gel. The nomenclature for the substitution is the same as was described in the Figure 1 legend. After visualization of Msh2∷HAp (Msh2), the membrane was reprobed with rabbit α-Kar2p polyclonal and α-rabbit IgG HRP antibodies (loading). (B) Representative example of overexpression assay. Overexpression was achieved by placement of the inducible GAL10 (GAL) promoter, a promoter that leads to repression during cultivation in glucose (off) but confers elevated expression in the presence of galactose and the absence of glucose (overexpressed) upstream of the MSH2 missense alleles. In addition, the GAL-MSH2 fusions were on plasmid DNA replicated via the high-copy 2μ origin of replication. The mismatch repair phenotypes of msh2Δ strains overexpressing the msh2 missense substitutions (example shown is C195R, GAL-msh2-C195R) were compared to those overexpressing the wild-type MSH2 gene (GAL-MSH2) and to those with no overexpression (vector). The mismatch repair efficiencies were determined qualitatively using the 5-fluororotic acid monohydrate (FOA) dinucleotide instability plate assays (Henderson and Petes 1992). (C) Stability residues cluster to four regions on the basis of MutS crystal structure modeling. The leftmost image is of the MutS homodimer structure with the relevant positions in black and enhanced with shading. The five structural domains are highlighted as follows: domain I (blue), domain II (green), domain III (yellow), domain IV (orange), and domain V (red). The four stability clusters are enlarged and labeled 1–4 in the numbered images on the right. The relevant amino acid with the codon number is given for yeast with the corresponding Thermus aquaticus, Taq, MutS residue in parentheses below. Images were generated by manipulating 1ewq.pdb (crystal structure Taq MutS homodimer complexed with a heteroduplex DNA molecule at 2.2 Å resolution) (Obmolova et al. 2000) using the Swiss PDB Viewer (version 3.7) (Guex et al. 1999) and POV Ray Tracer program (version 3.6). (1) The connection between domains I and II is shown with E194 and C195 highlighted. (2) The C345, T347, G350, A618, and D621 stability residues are clustered within a 5-Å radius of one another in a region in the central core of the protein in domain III (yellow). The linking helix connecting the DNA-binding domain (orange) with the ATPase domain (red) is emphasized. (3) The highly conserved stability residues P640, C716, R371, and G711 localize to a region where a surface-exposed helix containing R371 (domain III) makes contact with the ATPase-containing domain (domain V) to stabilize the region. (4) Three stability residues (L521, D524, and R542) are located near the DNA mismatch recognition site (domain IV) that becomes stabilized upon DNA binding and is adjacent to the connecting helix (Obmolova et al. 2000).

Figure 3.—

Mutations altering the mismatch repair complex. (A) Representative two-hybrid assay to detect mismatch repair subunit formation. The MATa yeast two-hybrid reporter strain was transformed with pGBD-C2 (negative control, C2), pGBD-MSH2 (positive control, Msh2), pGBD-msh2-P640L (Msh2^P640L), pGBD-msh2-H658Y (Msh2^H658Y), pGBD-msh2-G693D (Msh2^G693D), pGBD-msh2-G688D (Msh2^G688D), pGBD-msh2-T743I (Msh2^T743I), pGBD-msh2-G692S (Msh2^G692S), or pGBD-msh2-S742L (Msh2^S742L). Transformants were mated with (denoted with an ×) individual MATα yeast two-hybrid reporter strains harboring pGAD-MSH6 (Msh6), pGAD-MSH3 (Msh3), pGAD-MLH1 (Mlh1), pGAD-PMS1 (Pms1), pGAD-EXO1 (Exo1), or pGAD-POL30 (Pol30). Diploid cultures were serially diluted and spotted onto agar plates and allowed to grow for 2 days at 30°. Medium lacking leucine and tryptophan (–LEU –TRP) selects for diploids harboring both pGAD and pGBD constructs (Control). Growth on selective medium also lacking histidine (–LEU –TRP –HIS) indicates a two-hybrid interaction (Interaction). (B) Sample immunoblot to detect decreased levels of GBD-Msh2 fusion proteins. The positive control included the wild-type GBD-Msh2-HA protein extract from a strain expressing the fusion from pGBD-MSH2 (Msh2). The no-epitope control extracts were from a strain harboring the pGBD-C2 vector (C2). The msh2 alleles were expressed from a plasmid identical to pGBD-MSH2 except for the relevant encoded missense mutation. The denoted change is marked in superscript. The nomenclature for the substitution is the same as was described in the Figure 1 legend. After visualization of GBD-Msh2∷HAp (GBD-Msh2), the membrane was reprobed with rabbit α-Kar2p polyclonal and α-rabbit IgG HRP antibodies (loading). (C–F) Clustering of amino acid substitution sites resulting exclusively in subunit formation defects. The relevant yeast amino acid with the codon number is given with the corresponding Taq MutS residue in parentheses below. The subunit positions are colored in black and enhanced with shading. The DNA-contacting subunit of the homodimer (the Msh6- or Msh3-like subunit of Taq MutS) is shown in paler colors. For example, domain I is dark blue for the Msh2-like subunit and pale blue for the Msh6/3p-like subunit, whereas domain V is red for the Msh2-like subunit and pink for the Msh6/3p-like subunit. Images were generated as described in the previous figure legend. (C) Domain V subunit interface. In domain V (red for the Msh2-like subunit), the residue corresponding to yeast S762 is S669 (black) in Taq MutS and is within 4 Å of H696 (gray) in the other dimer partner (pink). (D) DNA-binding region subunit interface. Domain I is a region where the two dimer partners come in close proximity upon DNA binding. C67 (R76 in Taq MutS, black) on the Msh2-like subunit (dark blue) is within 4 Å of Taq MutS V57 (gray) in the dimer partner (light blue). (D) Surface-exposed residue L457. The image is of the Taq MutS homodimer structure with L457 (Taq MutS L372) surface residue in black. (F) Putative domain V connector region and putative contact region with Mlh1p (MutL). H658 and M707 residues are highlighted in black.

Figure 4.—

The composite ATPase positions. (A) Mapping the position onto the composite ATPase domain. The ATPase domains of the Taq MutS homodimer are shown (red for the Msh2-like subunit; pink for the Msh6/3p-like subunit). The relevant yeast amino acid with the codon number is given with the corresponding Taq MutS residue in parentheses below. The ADP molecules are shown in blue. The positions targeted for alteration corresponding to the conserved consensus ATPase active site are highlighted in black. The putative ATPase positions are highlighted in gray. The Msh6/3p-like dimer partner is shown in pink. Images were made as described in the Figure 3 legend, except that the PDB file was 1fw6.pdb, the crystal structure of a Taq MutS–DNA–ADP ternary complex (Junop et al. 2001). (B) Consensus sites for ATPases with the corresponding positions analyzed in this work. Boldface residues indicate the consensus for all ATPases. Underlined residues have a corresponding missense variant analyzed in this article. The DELGRG sequence conservation encompassing the Walker B box is for MutS homologs.