Correcting Replication Errors Research Articles

High resolution deletion mapping reveals frequent allelic losses at the DNA mismatch repair loci hMLH1 and hMSH3 in non‐small cell lung cancer

To study the involvement of DNA mismatch repair genes in non-small cell lung cancer, matched normal and tumoral DNA samples from 31 patients were analyzed for both LOH and microsatellite instability with 34 markers at or linked to hMLH1(3p21),hMSH2(2p16), hMSH3(5q11-q13),hMSH6(2p16), hPMS1(2q32), and hPMS2 (7p22) loci. Chromosomal regions 3p21 and 5q11-q13 were found to be hemizygously deleted in 55% and 42% of the patients, respectively. Sixty five percent of the patients deleted at hMLH1 were also deleted at hMSH3. The shortest regions of overlap for 3p21 and 5q11-q13 deletions delimited by D3S1561/D3S1612 and D5S2107/D5S624, respectively, were restricted to genetic distances of only 1 cM. Currently, the hMLH1 (3p21) and hMSH3 (5q11-q13) genes are the only known candidates located within these regions. The mutational analysis of hMLH1 and hMSH3 in hemizygously deleted patients led to the detection of 2 new polymorphisms in hMSH3. The consequence of these allelic losses remains unclear, but the lack of inactivating mutation might explain that replication error, the hallmark of mismatch repair genes inactivation in cancer cells, was quasi-absent in tumors. We suggest that hMLH1 and hMSH3 genes could be involved in lung tumorigenesis through dosage effect in cellular functions other than replication error correction. Int. J. Cancer 77:173–180, 1998. © 1998 Wiley-Liss, Inc.

DNA excision repair pathways

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair proteins in knockout mice provides information on the roles of such factors in vivo and recent three-dimensional structures of several repair enzymes explain there detailed modes of action.

Processing of O6-methylguanine by mismatch correction in human cell extracts

Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutSα mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase δ and/or ϵ. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.

DNA-replication fidelity, mismatch repair and genome instability in cancer cells.

It has been suggested that an early event in the multistep progression of a normal cell to a tumor cell could be a defect that leads to an elevated mutation rate, thus providing a pool of mutants upon which selection could act to yield a tumor. Such a mutator phenotype could result from a defect in any of several DNA transactions, including those that determine the DNA replication error rate or the ability to correct replication errors. Recent evidence for the latter is the mutator phenotype observed in tumor cells of patients having a hereditary form of colon cancer. These patients have a germline mutation in genes required for post-replication DNA mismatch repair. A second mutation arises somatically, yielding a greatly elevated mutation rate due to an inability to correct DNA replication errors. This connection between cancer, DNA replication errors and defective mismatch repair is the subject of this review, wherein we consider the key steps and principles for high fidelity replication and how their perturbation results in genome instability.

3'-->5' exonucleases of DNA polymerases epsilon and delta correct base analog induced DNA replication errors on opposite DNA strands in Saccharomyces cerevisiae.

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC-->AT and AT-->GC transitions that are enhanced in DNA polymerase epsilon and delta 3'-->5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases delta and epsilon contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC-->AT and AT-->GC transitions in a Pol+, pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases epsilon and delta correct HAP-induced DNA replication errors on opposite DNA strands.

DNA-mismatch repair. The intricacies of eukaryotic spell-checking.

Recent work suggests that the eukaryotic system responsible for repairing DNA mismatches, and so correcting replication errors, is more complex than was thought; its multiple components have many cellular functions.

Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes. We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.

Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities.

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.

Involvement of Escherichia coli mismatch repair in DNA replication and recombination.

The idea that there might be a process capable of recognizing and repairing incorrect base pairs in double-stranded DNA (mismatch repair) was originally proposed to account for results obtained from the study of genetic recombination. In particular, it was suggested that the repair of mismatched bases in heteroduplex regions of DNA (i.e., regions formed by strand exchange between DNA molecules with similar but not identical sequences) could be involved in gene conversion (Holliday 1964) and could account for the phenomenon of high negative interference (White and Fox 1974). That mismatch repair in E. coli might be involved in correcting replication errors was suggested by the finding that mutants deficient in mismatch repair have a mutator phenotype (Nevers and Spatz 1975; Rydberg 1978; Glickman and Radman 1980). Perhaps to a large extent because of its apparent involvement in mutation avoidance, mismatch repair has recently been widely studied. We will describe here...