Dissociation of mismatch recognition and ATPase activity by hMSH2-hMSH3.

MSH2-MSH3 directs the repair of insertion/deletion loops of up to 13 nucleotides in vivo and in vitro. To examine the biochemical basis of this repair specificity, we characterized the mispair binding and ATPase activity of hMSH2-hMSH3. The ATPase was found to be regulated by a mismatch-stimulated ADP --> ATP exchange, which induces a conformational transition by the protein complex. We demonstrated strong binding of hMSH2-hMSH3 to an insertion/deletion loop containing 24 nucleotides that is incapable of provoking ADP --> ATP exchange, suggesting that mismatch recognition appears to be necessary but not sufficient to induce the intrinsic ATPase. These studies support the idea that hMSH2-hMSH3 functions as an adenosine nucleotide-regulated molecular switch that must be activated by mismatched nucleotides for classical mismatch repair to occur.     

hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes

Five MutS homologs (MSH), which form three heterodimeric protein complexes, have been identified in eukaryotes. While the human hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers operate primarily in mitotic mismatch repair (MMR), the biochemical function(s) of the meiosis-specific hMSH4-hMSH5 heterodimer is unknown. Here, we demonstrate that purified hMSH4-hMSH5 binds uniquely to Holliday Junctions. Holliday Junctions stimulate the hMSH4-hMSH5 ATP hydrolysis (ATPase) activity, which is controlled by Holliday Junction-provoked ADP-->ATP exchange. ATP binding by hMSH4-hMSH5 induces the formation of a hydrolysis-independent sliding clamp that dissociates from the Holliday Junction crossover region, embracing two homologous duplex DNA arms. Fundamental differences between hMSH2-hMSH6 and hMSH4-hMSH5 Holliday Junction recognition are detailed. Our results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.     

Interactions of Human hMSH2 with hMSH3 and hMSH2 with hMSH6: Examination of Mutations Found in Hereditary Nonpolyposis Colorectal Cancer

Mutations in the human mismatch repair protein hMSH2 have been found to cosegregate with hereditary nonpolyposis colorectal cancer (HNPCC). Previous biochemical and physical studies have shown that hMSH2 forms specific mispair binding complexes with hMSH3 and hMSH6. We have further characterized these protein interactions by mapping the contact regions within the hMSH2-hMSH3 and the hMSH2-hMSH6 heterodimers. We demonstrate that there are at least two distinct interaction regions of hMSH2 with hMSH3 and hMSH2 with hMSH6. Interestingly, the interaction regions of hMSH2 with either hMSH3 or hMSH6 are identical and there is a coordinated linear orientation of these regions. We examined several missense alterations of hMSH2 found in HNPCC kindreds that are contained within the consensus interaction regions. None of these missense mutations displayed a defect in protein-protein interaction. These data support the notion that these HNPCC-associated mutations may affect some other function of the heterodimeric complexes than simply the static interaction of hMSH2 with hMSH3 or hMSH2 with hMSH6.     

hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.     

Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6

The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ～8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.     

The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3

The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents. Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity. In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity. In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers. Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3. These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6.     

Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex

Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSalpha is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSalpha activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.     

hMSH4-hMSH5 adenosine nucleotide processing and interactions with homologous recombination machinery

We have previously demonstrated that the human heterodimeric meiosis-specific MutS homologs, hMSH4-hMSH5, bind uniquely to a Holliday Junction and its developmental progenitor (Snowden, T., Acharya, S., Butz, C., Berardini, M., and Fishel, R. (2004) Mol. Cell 15, 437-451). ATP binding by hMSH4-hMSH5 resulted in the formation of a sliding clamp that dissociated from the Holliday Junction crossover region embracing two duplex DNA arms. The loading of multiple hMSH4-hMSH5 sliding clamps was anticipated to stabilize the interaction between parental chromosomes during meiosis double-stranded break repair. Here we have identified the interaction region between the individual subunits of hMSH4-hMSH5 that are likely involved in clamp formation and show that each subunit of the heterodimer binds ATP. We have determined that ADP-->ATP exchange is uniquely provoked by Holliday Junction recognition. Moreover, the hydrolysis of ATP by hMSH4-hMSH5 appears to occur after the complex transits the open ends of model Holliday Junction oligonucleotides. Finally, we have identified several components of the double-stranded break repair machinery that strongly interact with hMSH4-hMSH5. These results further underline the function(s) and interactors of hMSH4-hMSH5 that ensure accurate chromosomal repair and segregation during meiosis.     

Functional characterization of two human MutY homolog (hMYH) missense mutations (R227W and V232F) that lie within the putative hMSH6 binding domain and are associated with hMYH polyposis

The base excision repair DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated into DNA opposite guanine or 7,8-dihydro-8-oxo-guanine (8-oxoG), thereby preventing G:C to T:A mutations. Biallelic germline mutations in the human MYH gene predispose individuals to multiple colorectal adenomas and carcinoma. We have recently demonstrated that hMYH interacts with the mismatch repair protein hMSH6, and that the hMSH2/hMSH6 (hMutSα) heterodimer stimulates hMYH activity. Here, we characterize the functional effect of two missense mutations (R227W and V232F) associated with hMYH polyposis that lie within, or adjacent to, the putative hMSH6 binding domain. Neither missense mutation affects the physical interaction between hMYH and hMSH6. However, hMYH(R227W) has a severe defect in A/8-oxoG binding and glycosylase activities, while hMYH(V232F) has reduced A/8-oxoG binding and glycosylase activities. The glycosylase activity of the V232F mutant can be partially stimulated by hMutSα but cannot be restored to the wild-type level. Both mutants also fail to complement mutY-deficiency in Escherichia coli. These data define the pathogenic mechanisms underlying two further hMYH polyposis-associated mutations.     

hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSalpha.

In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutSalpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160). Recombinant hMutSalpha bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower. In the presence of ATP, hMutSalpha dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine. Surprisingly, this reaction required only ATP binding, not hydrolysis. The ATPase activity of hMutSalpha variants carrying the Lys-->Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts. The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory. We conclude that although the ATPase activity of hMutSalpha is dispensible for mismatch binding, it is required for mismatch correction.     

The interaction of the human MutL homologues in hereditary nonpolyposis colon cancer

Germline mutations in two human mismatch repair (MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70% of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to copurify with another MMR protein hPMS2 as a heterodimer, their function in MMR is unknown. In this study, we have identified the physical interaction regions of both hMLH1 with hPMS2. We then examined the effects of hMLH1 missense alterations found in HNPCC kindreds for their interaction with hPMS2. Four of these missense alterations (L574P, K616Delta, R659P, and A681T) displayed >95% reduction in binding to hPMS2. Two additional missense alterations (K618A and K618T) displayed a >85% reduction in binding to hPMS2, whereas three missense alterations (S44F, V506A, and E578G) displayed 25-65% reduction in binding to hPMS2. Interestingly, two HNPCC missense alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data confirm that functional deficiencies in the interaction of hMLH1 with hPMS2 are associated with HNPCC as well as suggest that other unknown functional alteration of the human MutL homologues may lead to tumorigenesis in HNPCC kindreds.     

Mutation in the magnesium binding site of hMSH6 disables the hMutSalpha sliding clamp from translocating along DNA

In human cells, binding of base/base mismatches and small insertion/deletion loops is mediated by hMutSalpha, a heterodimer of hMSH2 and hMSH6. In the presence of ATP and magnesium, hMutSalpha dissociates from the mismatch by following the DNA contour in the form of a sliding clamp. This process is enabled by a conformational change of the heterodimer, which is driven by the binding of ATP and magnesium in the Walker type A and B motifs of the polypeptides, respectively. We show that a purified recombinant hMutSalpha variant, hMutSalpha 6DV, which contains an aspartate to valine substitution in the Walker type B motif of the hMSH6 subunit, fails to undergo the conformational change compatible with translocation. Instead, its direct dissociation from the mismatch-containing DNA substrate in the presence of ATP and magnesium precludes the assembly of a functional mismatch repair complex. The "translocation-prone" conformation of wild type hMutSalpha could be observed solely under conditions that favor hydrolysis of the nucleotide and mismatch repair in vitro. Thus, whereas magnesium could be substituted with manganese, ATP could not be replaced with its slowly or nonhydrolyzable homologues ATP-gammaS or AMPPNP, respectively. The finding that ATP induces different conformational changes in hMutSalpha in the presence and in the absence of magnesium helps explain the functional differences between hMutSalpha variants incapable of binding ATP as compared with those unable to bind the metal ion.     

A quantitative model of nucleosome dynamics

The expression, replication and repair of eukaryotic genomes require the fundamental organizing unit of chromatin, the nucleosome, to be unwrapped and disassembled. We have developed a quantitative model of nucleosome dynamics which provides a fundamental understanding of these DNA processes. We calibrated this model using results from high precision single molecule nucleosome unzipping experiments, and then tested its predictions for experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex hMSH2-hMSH6. We found that this calibrated model quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate DNA sequences and four distinct histone modification states. In addition, this model provides mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone modifications on this disassembly reaction. This model''s precise agreement with current experiments suggests that it can be applied more generally to provide important mechanistic understanding of the numerous nucleosome alterations that occur during DNA processing.     

Mutator phenotypes of common polymorphisms and missense mutations in MSH2.

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2 [1], the human homologue of the Escherichia coli MutS gene. These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair. However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins. Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain. Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates. Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor. Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s). The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.     

The role of mismatched nucleotides in activating the hMSH2-hMSH6 molecular switch

We have previously shown that hMSH2-hMSH6 contains an intrinsic ATPase which is activated by mismatch-provoked ADP-->ATP exchange that coordinately induces the formation of a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone (1,2). These studies suggested that mismatch repair could be propagated by a signaling event transduced via diffusion of ATP-bound hMSH2-hMSH6 molecular switches to the DNA repair machinery. The Molecular Switch model (Fishel, R. (1998) Genes Dev. 12, 2096-2101) is considerably different than the Hydrolysis-Driven Translocation model (Blackwell, L. J., Martik, D., Bjornson, K. P., Bjornson, E. S., and Modrich, P. (1998) J. Biol. Chem. 273, 32055-32062) and makes additional testable predictions beyond the demonstration of hydrolysis-independent diffusion (Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., and Fishel, R. (1999) Mol. Cell 3, 255-261): (i) individual mismatch-provoked ADP-->ATP exchange should be unique and rate-limiting, and (ii) the k(cat x DNA) for the DNA-stimulated ATPase activity should decrease with increasing chain length. Here we have examined hMSH2-hMSH6 affinity and ATPase stimulatory activity for several DNA substrates containing mispaired nucleotides as well as the chain length dependence of a defined mismatch under physiological conditions. We find that the results are most consistent with the predictions of the Molecular Switch model.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

Mismatch recognition and DNA-dependent stimulation of the ATPase activity of hMutSalpha is abolished by a single mutation in the hMSH6 subunit

The most abundant mismatch binding factor in human cells, hMutSalpha, is a heterodimer of hMSH2 and hMSH6, two homologues of the bacterial MutS protein. The C-terminal portions of all MutS homologues contain an ATP binding motif and are highly conserved throughout evolution. Although the N termini are generally divergent, they too contain short conserved sequence elements. A phenylalanine --> alanine substitution within one such motif, GXFY(X)(5)DA, has been shown to abolish the mismatch binding activity of the MutS protein of Thermus aquaticus (Malkov, V. A., Biswas, I., Camerini-Otero, R. D., and Hsieh, P. (1997) J. Biol. Chem. 272, 23811-23817). We introduced an identical mutation into one or both subunits of hMutSalpha. The Phe --> Ala substitution in hMSH2 had no effect on the biological activity of the heterodimer. In contrast, the in vitro mismatch binding and mismatch repair functions of hMutSalpha were severely attenuated when the hMSH6 subunit was mutated. Moreover, this variant heterodimer also displayed a general DNA binding defect. Correspondingly, its ATPase activity could not be stimulated by either heteroduplex or homoduplex DNA. Thus the N-terminal portion of hMSH6 appears to impart on hMutSalpha not only the specificity for recognition and binding of mismatched substrates but also the ability to bind to homoduplex DNA.     

Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense mutations in ATP13A2 associated with early-onset forms of parkinsonism.     

Formation of hMSH4-hMSH5 heterocomplex is a prerequisite for subsequent GPS2 recruitment

Increasing evidence suggests that components of the DNA mismatch repair (MMR) pathway play multifunctional roles beyond the scope of mismatch correction, including the modulation of cellular responses to DNA damage and homologous recombination. The heterocomplex consisting of MutS homologous proteins, hMSH4 and hMSH5, is believed to play essential roles in meiotic DNA repair particularly during the process of meiotic homologous recombination (HR). In order to gain a better understanding of the mechanistic basis underlying the roles of these two human MutS proteins, we have identified G-protein pathway suppressor 2 (GPS2) (i.e., an integral component of a deacetylase complex) as an interacting protein partner specifically for the hMSH4-hMSH5 heterocomplex. The interaction with GPS2 is entirely dependent on the physical association between hMSH4 and hMSH5, as disruption of the interaction between hMSH4 and hMSH5 completely abolishes GPS2 recruitment. Our analysis further indicates that the association with GPS2 is mediated through the interface of hMSH4-hMSH5 complex and the N-terminal region of GPS2. Moreover, these three proteins interact in human cells, and analysis of microarray data suggested a coordinated expression pattern of these genes during the onset of meiosis. Together, the results of our present study suggest that the GPS2-associated deacetylase complex might function in concert with hMSH4-hMSH5 during the process of homologous recombination.     

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae

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.