A mismatch-binding heterodimer of hMSH2 and a 160-kilodalton polypeptide has been isolated from HeLa cells by virtue of its ability to restore mismatch repair to nuclear extracts of hMSH2-deficient LoVo colorectal tumor cells. This heterodimer, designated hMutS alpha, also restores mismatch repair to extracts of alkylation-tolerant MT1 lymphoblastoid cells and HCT-15 colorectal tumor cells, which are selectively defective in the repair of base-base and single-nucleotide insertion-deletion mismatches. Because HOT-15 cells appear to be free of hMSH2 mutations, this selective repair defect is likely a result of a deficiency of the hMutS alpha 160-kilodalton subunit, and mutations in the corresponding gene may confer hypermutability and cancer predisposition.

Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSIpositive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism.Germ-line defects in DNA mismatch repair (MMR) genes account for the inherited familial cancer syndrome of hereditary nonpolyposis colon cancers in which affected individuals show accelerated development of cancers of the proximal colon, endometrium, and stomach (1-5). These cancers typically demonstrate inactivation of the residual wild-type MMR allele inherited opposite the germ-line mutant (1-6), absence of DNA MMR activity in in vitro assays (7,8), and acquisition of an in vivo mutator phenotype showing up to 1,000-fold increased gene mutation rates (9, 10). Additionally, these cancers display an associated instability of genomic microsatellite sequences (MSI) (1-5). MSI is similarly found in approximately 15-20% of sporadic colon cancers that arise in individuals without any family history of colon cancer (1-5). Similarly to hereditary nonpolyposis colon cancer-associated colon cancers, sporadic MSI colon cancers arise predominantly in the proximal colon and show a high rate of frameshift mutations at a mutational hotspot in the transforming growth factor-␤ type II receptor tumor suppressor gene (1, 11). Familial and sporadic MSI colon cancers thus appear to share a common carcinogenic pathway. In this regard, previous studies from our group established that MMR gene inactivation via somatic mutation was the cause of some cases of sporadic MSI colon cancers (12). However, unexpectedly, in many spo...

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Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSa recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between 04-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by transdiamminedichloroplatinum(H) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(ll) adducts by MutSa may be involved in the cytotoxic action of this chemotherapeutic agent.

DNA mismatch repair is central to the maintenance of genomic stability. It is initiated by the recognition of base-base mismatches and insertion͞deletion loops by the family of MutS proteins. Subsequently, ATP induces a unique conformational change in the MutS-mismatch complex but not in the MutS-homoduplex complex that sets off the cascade of events that leads to repair. To gain insight into the mechanism by which MutS discriminates between mismatch and homoduplex DNA, we have examined the conformations of specific and nonspecific MutS-DNA complexes by using atomic force microscopy. Interestingly, MutS-DNA complexes exhibit a single population of conformations, in which the DNA is bent at homoduplex sites, but two populations of conformations, bent and unbent, at mismatch sites. These results suggest that the specific recognition complex is one in which the DNA is unbent. Combining our results with existing biochemical and crystallographic data leads us to propose that MutS: (i) binds to DNA nonspecifically and bends it in search of a mismatch; (ii) on specific recognition of a mismatch, undergoes a conformational change to an initial recognition complex in which the DNA is kinked, with interactions similar to those in the published crystal structures; and (iii) finally undergoes a further conformational change to the ultimate recognition complex in which the DNA is unbent. Our results provide a structural explanation for the long-standing question of how MutS achieves mismatch repair specificity. D NA mismatch repair (MMR) is a highly conserved repair pathway targeting mismatched bases that arise through DNA replication errors and during homologous recombination (1-3). Inactivation of MMR genes results in a significant increase in the spontaneous mutation rate and, in humans, a predisposition to cancer (4). Escherichia coli provides the best-understood MMR system and serves as a prototype for the more complicated but homologous eukaryotic systems (5). In E. coli, the proteins MutS, MutL, and MutH are responsible for the initiation of MMR (6). MutS and MutL function as dimers and have intrinsic ATPase activities that are essential for MMR (7,8). MMR is initiated by the binding of MutS to either a mismatch or a short insertion͞deletion loop (IDL). Subsequently, ATP induces a conformational change in the MutS-mismatch complex and promotes its interaction with MutL. Assembly of the MutS-MutL-heteroduplex complex activates the endonuclease activity of MutH, which incises the newly synthesized (unmethylated) strand at a d(GATC) site. This incision confers strand specificity of MMR, directing repair exclusively to the newly synthesized strand containing the error. Excision repair completes the process.Crystal structures of E. coli and Thermus aquaticus (Taq) MutS dimers complexed with a G͞T base-base mismatch and a 1T-bulge, respectively, shed light on the structural components of mismatch recognition (9-11). Specific interactions include an aromatic ring stack of a conserved phenylalanine (Phe-39 in Taq or Phe-36 in...

In contrast to parental A2780 ovarian tumor cells, extracts of one doxorubicin-resistant and two independent cis-diamminedichloroplatinum(II)-resistant derivatives are defective in strand-specific mismatch repair. The repair defect of the three hypermutable, drug-resistant cell lines is only evident when the strand break that directs the reaction is located 3 to the mismatch, and in each case repair is restored to extracts by addition of purified MutL␣ heterodimer. As judged by immunological assay, drug resistance is associated with the virtual absence of the MutL␣ MLH1 subunit and greatly reduced levels of the PMS2 subunit. These findings implicate a functional mismatch repair system in the cytotoxic effects of these antitumor drugs and may have ramifications for their clinical application.

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutS␣ (hMSH2⅐hMSH6) and hMutS␤ (hMSH2⅐hMSH3). In HL-60 extracts the hMutS␣ to hMutS␤ ratio is roughly 6:1, whereas in methotrexateresistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of basebase mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutS␣ and hMutS␤ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutS␣ restores basebase mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.

Nucleotide excision repair and the long-patch mismatch repair systems correct abnormal DNA structures arising from DNA damage and replication errors, respectively. DNA synthesis past a damaged base (translesion replication) often causes misincorporation at the lesion site. In addition, mismatches are hot spots for DNA damage because of increased susceptibility of unpaired bases to chemical modification. We call such a DNA lesion, that is, a base damage superimposed on a mismatch, a compound lesion. To learn about the processing of compound lesions by human cells, synthetic compound lesions containing UV photoproducts or cisplatin 1,2-d(GpG) intrastrand cross-link and mismatch were tested for binding to the human mismatch recognition complex hMutS␣ and for excision by the human excision nuclease. No functional overlap between excision repair and mismatch repair was observed. The presence of a thymine dimer or a cisplatin diadduct in the context of a G-T mismatch reduced the affinity of hMutS␣ for the mismatch. In contrast, the damaged bases in these compound lesions were excised three-to fourfold faster than simple lesions by the human excision nuclease, regardless of the presence of hMutS␣ in the reaction. These results provide a new perspective on how excision repair, a cellular defense system for maintaining genomic integrity, can fix mutations under certain circumstances.Mismatches in DNA resulting from replication errors, and base damage caused by physical (UV light) and chemical (polyaromatic hydrocarbons) agents, are responsible for the majority of human cancers (18). Although certain mismatches and base lesions can be eliminated from DNA by the base excision repair pathway initiated by glycosylases with narrow substrate ranges, in human cells there exists a general mismatch repair system and a general damage repair system of wide substrate range. The mismatch repair system removes the mismatched base as a nucleotide (44), and the excision repair system excises the damaged base(s) in an oligonucleotide (52, 67).The general mismatch repair system (long-patch mismatch repair) corrects all eight single-base mismatches as well as small insertion sequence loops with comparable efficiencies (31, 44). The general nucleotide excision repair (excision repair) system (8) not only is the sole repair pathway for bulky lesions such as thymine dimers (TϽϾT) and cisplatin-guanine adducts but also repairs a wide variety of nonbulky lesions such as O 6 -methylguanine (O 6 -meG) at physiologically relevant rates (52). Thus, it appears that both repair systems recognize many dissimilar non-B DNA forms rather than a specific lesion structure.Given the wide substrate ranges of both systems, it is not unreasonable to expect overlaps between the two substrate spectra, or that one repair system may facilitate the function of the other. Indeed, it has been shown that the excision repair system recognizes mismatches and removes the mismatched base in a manner identical to the removal of damaged bases (27). Since a DNA lesion, by de...