Expression of long-patch and short-patch DNA mismatch repair proteins in the embryonic and adult mammalian brain

Expression of the DNA mismatch repair (MMR) pathway was examined in the adult and developing rat brain. Rat homologues of human GTBP and MSH2, which are essential components of the post-replicative DNA MMR system, were identified in nuclear extracts from the adult and developing rat brain. Developmental studies showed that both GTBP and MSH2 levels were higher in nuclei isolated from the embryonic brain (day 16) than adult brain. However, this difference was not as dramatic as the difference in the number of proliferating cells. Levels of thymine DNA glycosylase (TDG), the enzyme which catalyzes the first step in short patch G:T mismatch repair, were also decreased in adult compared to embryonic brain. In the adult brain, MMR proteins were elevated in nuclear extracts enriched for neuronal nuclei. These results suggest that adult brain cells have the capacity to carry out DNA mismatch repair, in spite of a lack of ongoing DNA replication.     

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.     

Usefulness of electron beam computed tomography scanning for distinguishing ischemic from nonischemic cardiomyopathy

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.     

Differential induction of c-Jun NH2-terminal kinase and c-Abl kinase in DNA mismatch repair-proficient and -deficient cells exposed to cisplatin

The c-Abl nonreceptor tyrosine kinase and the c-Jun NH2-terminal kinase (JNK/stress-activated protein kinase) are activated during the injury response to the DNA-damaging agent cisplatin. Loss of DNA mismatch repair activity results in resistance to cisplatin in human cancer cells, suggesting that the mismatch repair proteins function as a detector for cisplatin DNA adducts. To identify signaling pathways activated by this detector, we investigated the effect of the loss of DNA mismatch repair function on the ability of cisplatin to activate the JNK and c-Abl kinases. The results demonstrate that cisplatin activates JNK kinase 3.8 +/- 0.2-fold more efficiently in DNA mismatch repair-proficient than repair-deficient cells, and that activation of c-Abl is completely absent in the DNA mismatch repair-deficient cells. Furthermore, the results show that cisplatin-induced activation of JNK occurs through a stress-activated protein kinase/extracellular signal-regulated kinase kinase 1-independent mechanism. We conclude that activation of JNK and c-Abl by cisplatin is in part dependent upon the integrity of DNA mismatch repair function, suggesting that these kinases are part of the signal transduction pathway activated when mismatch repair proteins recognize cisplatin adducts in DNA.     

Mismatch repair protein

A DNA mismatch repair system exists that repairs mispaired bases formed during DNA replication and genetic recombination. Genetic defects in this mismatch repair system are known to increase the rate of spontaneous mutation in Escherichia coli. Some cases of inherited cancer are associated with inherited defects of mismatch repair genes, showing the importance of the mismatch repair system in maintenance of genetic stability and avoidance of cancer susceptibility. This review focused on what is known about the mechanisms of mismatch repair in human cells and the relationship between defects in mismatch repair and carcinogenesis.     

Eukaryotic mismatch repair: an update

The discovery that mutations in mismatch repair genes segregate with hereditary nonpolyposis colon cancer has awakened a great deal of interest in the study of the process of postreplicative mismatch repair. The characterisation of the principal players involved in this important metabolic pathway has been greatly facilitated by the amino acid sequence conservation among functional homologues of bacteria, yeast and mammals. The phenotypes of mismatch repair deficient mutants are also similar in many ways. In humans, mismatch repair malfunction demonstrates itself in the form of a mutator phenotype of the affected cells, an instability of microsatellite sequences and increased levels of somatic recombination. Moreover, mismatch repair deficient cells display also varying levels of tolerance to DNA damaging agents and are thought to be involved in the cell killing mediated by these agents. This article discusses some recent developments in this fast-moving field.     

MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2

The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved. In Escherichia coli, DNA mismatches are recognized by the MutS protein. Homologues of the E. coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals. Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro. However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP. It has been reported that S. cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2. We show here that, as in human cells, the S. cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.     

Resistance to cytotoxic drugs in DNA mismatch repair-deficient cells

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.     

Drug-induced sleep disorders]

Mismatch repair genes are involved in increasing the fidelity of replication by specific repair of DNA polymerase incorporation errors. In Escherichia coli, the best studied mismatch repair (MMR) pathway is the methyl-directed long patch repair system which is mediated by three gene products; MutS, MutL and MutH. These are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding regions of MMR genes result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair.     

Mutation of a mutL homolog in hereditary colon cancer

Some cases of hereditary nonpolyposis colorectal cancer (HNPCC) are due to alterations in a mutS-related mismatch repair gene. A search of a large database of expressed sequence tags derived from random complementary DNA clones revealed three additional human mismatch repair genes, all related to the bacterial mutL gene. One of these genes (hMLH1) resides on chromosome 3p21, within 1 centimorgan of markers previously linked to cancer susceptibility in HNPCC kindreds. Mutations of hMLH1 that would disrupt the gene product were identified in such kindreds, demonstrating that this gene is responsible for the disease. These results suggest that defects in any of several mismatch repair genes can cause HNPCC.     

Functional analysis of human MutSalpha and MutSbeta complexes in yeast

Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of these human protein complexes in yeast. We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain. Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA. The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast. Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding. The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.     

Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer

The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC). On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref. 3). Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p21.3-23. We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family.     

Effects of the human immunodeficiency virus type 1 Rev protein on reporter gene and host T-cell gene expression

The mechanism of DNA mismatch repair has been modeled upon biochemical studies of the E. coli DNA adenine methylation-instructed pathway where the initial recognition of mismatched nucleotides is performed by the MutS protein. MutS homologs (MSH) have been identified based on a highly conserved region containing a Walker-A adenine nucleotide binding motif. Here we show that adenine nucleotide binding and hydrolysis by the human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. The hMSH2-hMSH6 complex is ON (binds mismatched nucleotides) in the ADP-bound form and OFF in the ATP-bound form. These results suggest a new model for the function of MutS proteins during mismatch repair in which the switch determines the timing of downstream events.     

DNA-dependent activation of the hMutSalpha ATPase

ATP hydrolysis by MutS homologs is required for function of these proteins in mismatch repair. However, the function of ATP hydrolysis in the repair reaction is controversial. In this paper we describe a steady-state kinetic analysis of the DNA-activated ATPase of human MutSalpha. Comparison of salt concentration effects on mismatch repair and mismatch-provoked excision in HeLa nuclear extracts with salt effects on the DNA-activated ATPase suggests that ATP hydrolysis by MutSalpha is involved in the rate determining step in the repair pathway. While the ATPase is activated by homoduplex and heteroduplex DNA, the half-maximal concentration for activation by heteroduplex DNA is significantly lower under physiological salt concentrations. Furthermore, at optimal salt concentration, heteroduplex DNA increases the kcat for ATP hydrolysis to a greater extent than does homoduplex DNA. We also demonstrate that the degree of ATPase activation is dependent on DNA chain length, with the kcat for hydrolysis increasing significantly with chain length of the DNA cofactor. These results are discussed in terms of the translocation (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997) EMBO J. 16, 4467-4476) and the molecular switch (Gradia, S., Acharya, S., and Fishel, R. (1997) Cell 91, 995-1005) models that invoke distinct roles for ATP hydrolysis in MutS homolog function.     

Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.     

Characterization of a protein recognizing minor groove binders-damaged DNA

By using electromobility shift assay (EMSA), we have identified a protein able to recognize the DNA only if it was previously reacted with minor groove binders. This protein binds with very high affinity AT containing DNA treated with minor groove binders such as distamycin A, Hoechst 33258 and 33342, CC-1065 and ethidium bromide minor groove intercalator, but not with major groove binders such as quinacrine mustard, cisplatin or melphalan, or with topoisomerase I inhibitor camptothecin or topoisomerase II inhibitor doxorubicin. This protein was found to be present in different extracts of human, murine and hamster cells, with the human protein which appears to have a molecular weight slightly lower than that of the other species. This protein was found to be expressed both in cancer and normal tissues. By using molecular ultrafiltration techniques as well as southwestern analysis it was estimated that the apparent molecular weight is close to 100 kDa. We can exclude an identity between this protein and other proteins, with a similar molecular weight previously reported to be involved in DNA damage recognition/repair, such as topoisomerase I, mismatch repair activities such as the prokaryotic MutS protein and its human homologue hMSH2 or proteins of the nucleotide excision repair system such as ERCC1, -2, -3 and -4.