In this study, a prospective genetic screen in yeast was developed to identify codon changes in MLH1
causing loss-of-MMR function. A key feature of this screen was the use of functional human-yeast hybrid genes (51
), which allowed isolation of mutations in the ATPase domain of the human MLH1p. In earlier studies, prospective genetic screens have been performed in E.coli
) and yeast (71
) in order to investigate the structure and function of the native MMR proteins in those organisms. However, a prospective genetic screen for mutations specific to human MLH1
coding sequences has not been previously reported. In total, we have assembled functional information on 162 amino acid substitutions in the ATPase domain of MLH1p. The data consists of 117 replacements that impair MMR function, seven functionally silent alterations and the effects of all amino acid substitutions at residues K43 and S44 on protein function. Importantly, previously reported HNPCC-associated alterations [see MLH1
mutational databases and other reports (64
)] exhibited considerable overlap with the loss-of-MMR function mutations reported here. Of 33 distinct HNPCC-associated substitutions which have been reported in the N-terminal end of human MLH1p, 10 identical alterations were isolated in our prospective screen (see Figure ). The novel mutations identified in this study may aid in the interpretation of HNPCC genetic tests if the same variation is observed clinically.
This study describes a novel method for the identification of human MMR gene sequence alterations that impair function of the encoded protein in MMR. If such variants occur in humans, individuals harboring such alleles may have an elevated mutation rate and a predisposition to develop cancer. The method employs the yeast S.cerevisiae
, which has been used previously for the identification of mutant MMR genes. For example, Jeyaprakash et al
) used genetic complementation experiments and then direct cloning and DNA sequencing to ascertain the identity of the mutant gene in yeast strains with preexisting defects in microsatellite stability. More recent reports describe global mutagenesis of yeast, selection of yeast strains for those having alterations in MMR gene activity followed by cloning and DNA sequencing (71
). It should be noted that these studies were focused on finding variants of the native yeast proteins. Indeed, if reported at all, expression of the human MMR proteins in yeast has either no detectable biological activity (MSH2, MSH3 and MSH6) or induces a dominant negative mutator phenotype (MLH1 and the MSH2–MSH6 heterodimers) (47
). Previous studies have attempted to bypass these impediments by using, e.g. an hMSH2
fusion gene to screen for stop codons in the hMSH2
coding sequence or assays based on gain or loss of the dominant mutator phenotype (47
). However, these assays do not measure the native biological activity of the protein. The approach described in this study, employing hybrid human-yeast MMR proteins that are functional in MMR (51
), has allowed functional analyses of substitutions in the native human MMR gene sequence in yeast.
In this investigation, 27 codon alterations were isolated on two or more occasions (see Tables and ). Interestingly, only 4 of these 27 duplicates could have been due to cloning of the same amplified mutant from a single PCR fragment pool (data not shown). Instead, the majority of duplications were isolated in different gap repair screens using different PCR fragment pools and therefore represent independent generation of the same mutation. Thus, an important source of duplicity must be that (i) certain nucleotides are more prone to mutagenesis, i.e. ‘mutational hotspots’ (although two different polymerases and PCR amplification conditions were employed; see Materials and Methods) or (ii) the screen is reaching its limits in terms of the number of loss-of-function mutations possible. These possibilities are not mutually exclusive and the duplications may, in fact, result from a combination of these factors.
The results of this investigation raise an important question. How many loss-of-MMR function mutations might be expected in the human MLH1 region that was mutagenized? To estimate this number, we compared the experimental results to all possible single nucleotide missense codons at human codons 41–86 and 77–134. This analysis was restricted to the human portion of each hybrid since the crossover site sites between yeast sequences in the vector and mutagenized fragment during in vivo gap repair will vary slightly between clones. The first estimate was based on the number of possible termination codons actually isolated in the prospective screen (Table ). For hybrids MLH1_h(41-86) and MLH1-h(77-134), 5 of 16 (31%) and 5 of 21 (23%) of possible stop codons, respectively, were isolated. A second estimate is based on the results obtained by making all possible amino acid substitutions at human residues K43 and S44. At position 43, six different single base substitution codon changes (K43E, K43I, K43N, K43Q, K43R and K43T) are possible. Three of these exhibit 2-fold or greater loss-of-MMR function in vivo (Figure ; K43E, K43I and K43T). Only a K43I codon was isolated in the prospective screen. At position 44, six different single base substitution codon changes (S44A, S44C, S44F, S44P, S44T and S44Y) are possible. Five of these confer loss-of-MMR function (Figure ; S44C, S44F, S44P, S44T and S44Y), and one (S44F) was isolated in the prospective screen (Table ). Thus, at human codons K43 and S44, 33% (1 of 3) and 20% (1 of 5), respectively, of all single base substitution mutations were isolated. Cumulatively, this analysis suggests that we have identified ~25% of all single base substitution missense mutations in this region, implying that the total number of MLH1-inactivating amino acid substitutions in the region of MLH1 subjected to mutagenesis is ~460.
The majority of amino acid substitutions that conferred a loss-of-MMR function were isolated at highly-conserved residues (Figure ). Only 3 of the 117 substitutions that conferred loss-of-MMR function (hMLH1 K52E, K118I and P139H) occurred as the native amino acid at the equivalent position in any MLH1p from nine other species. In contrast, five of seven substitutions that exhibited little-to-no loss-of-MMR function (Table ) occurred naturally at the equivalent position in other species. There were several amino acid substitutions whose functional consequence may not have been predicted based on either the variation in nature or the biochemical properties of the alternate residues. For example, we found the biochemically conservative alterations hMLH1p D38E (both acidic), S46T (both uncharged polar) and N64S (both uncharged polar) resulted in loss-of-MMR function. Some alterations that may have been expected to confer detrimental effects on MMR function gave little-to-no loss of function. Thus, the substitutions I72T, K43C and K43G, which considerably alter the charge, polarity and size of the residue, did not substantially impair MMR function. Clearly, the functional consequence of any substitution requires direct experimental evaluation. This point is particularly relevant for the interpretation of HNPCC genetic tests, which often reveal novel missense codons in MLH1 and other MMR genes.
As summarized in Figure , amino acid substitutions causing loss-of-MMR function were identified at 61 residues throughout the N-terminal ATPase domain of MLH1p. Previous structural models of E.coli
) and biochemical studies using yeast and human MLH1p (37
) provide a foundation for understanding as to why substitutions at certain residues may impair MMR function. Three substitutions causing loss-of-MMR function were identified at the residue corresponding to hMLH1p N38, which helps to position a centrally located Mg2+
ion presumed to be important for ATP binding (38
). The amino acid substitutions identified (corresponding to hMLH1p N38D, N38S and N38T) may either abolish Mg2+
interactions or alter the placement of this critical ion. Substitutions were also identified at residues corresponding to hMLH1p D63, T82, S83, F99 and R100, which appear to have a direct role in ATP binding (38
). These substitutions are likely to perturb ATP binding and/or hydrolysis and, thus, prevent the conformational changes in MLH1p which are predicted to signal downstream MMR events. Most of the substitutions causing loss-of-MMR function were localized in the region corresponding to hMLH1p 41 through 86, which make up a large portion of the ATP-binding pocket and ‘ATP lid’ (41
). Although this region is certainly important for DNA MMR, we suspect that many mutations were localized here because, as depicted in Figure B, this portion of the mutant MLH1
gene originates solely from recombination with a mutagenized PCR fragment. A mutational analysis of other regions of MLH1
will require different gapped vector DNAs and the use of additional human-yeast hybrid MLH1
Based on the crystal structure of E.coli
MutL, human MLH1p residues K43 and S44 are expected to lie adjacent to helix αA within a conserved ATP-binding motif (motif I)(41
). It has been predicted that mutations in and around helix Aα dislocate a conserved glutamic acid residue, corresponding to hMLH1p E34, which is important for ATP binding and hydrolysis (37
). Specific biochemical functions have not been assigned to K43 or S44, but as shown in this investigation alterations in the size, charge and polarity of these residues result in distinct functional consequences for MLH1p. Previous investigations using the native yeast MLH1p have shown that substitutions mimicking a human HNPCC alteration (human MLH1p S44F) (34
) result in a loss-of-MMR function in vivo
). We confirmed this finding using the human-yeast hybrid gene MLH1_h(41-86) and showed that all amino acid substitutions except S44A impaired MMR function. At position 44, it appears that the small, uncharged side chains on serine and alanine are critical for proper MLH1p structure and/or function. At position K43, the systematic substitution of all possible amino acids resulted in a variety of functional consequences. In general, the introduction of amino acids with bulky and/or hydrophobic side chains tended to result in loss-of-MMR activity, while the introduction of amino acids with hydrophilic side chains were functionally tolerated. In addition, the results of this systematic analysis of all possible amino acid substitutions is consistent with our previous investigations, in which it was shown that substitutions in the human population can be either silent polymorphisms, inactivating mutations, or give rise to proteins that reduce the efficiency of MMR relative to the parental molecule (51
The goal of this investigation is to identify MLH1p amino acid substitutions which impair DNA MMR in vivo
. As noted, both human-yeast hybrid MLH1 proteins are functional in MMR but at a reduced efficiency compared to the native yeast protein. This is presumably due to the replacement of the yeast coding sequence with the conserved, but not identical, homologous human coding sequence. There is a formal possibility, therefore, that some of the missense codons we identified are specific to the hybrid protein and might not be observed when introduced to a native protein. Extrapolation of the results of this study to native proteins should consider the caveat that a subset of the variants may be specific to the hybrid molecule. At this time, it is not possible to conclude whether the missense mutations identified perturb other functions of MLH1p, such as its role in meiotic crossing-over (25
), and signaling of apoptosis in response to DNA damage (31
). However, based on the results of a recent investigation (80
), it appears that the majority of the mutations the ATPase domain that inactivate MMR also inactivate meiotic crossing-over. Further studies will be required to determine whether the ATPase activity of MLH1p is critical in other functions of the protein. Further studies will also be required to determine whether the substitutions have detrimental effects on the stability and/or solubility of MLH1p, protein–protein interactions and/or specific steps in ATP binding or hydrolysis (17
Understanding the functional consequence of missense codons in MLH1 is critical for increasing the utility of genetic tests for HNPCC. As described in this investigation, a rapid and biologically relevant strategy may be to accumulate functional information concerning putative disease-causing mutations in a prospective manner. Extending the prospective screen through additional portions of human MLH1 and other MMR genes could result in a comprehensive catalog of missense mutations that cause loss-of-MMR function.