mutL Gene Research Articles

DNA Loop Repair by Escherichia coli Cell Extracts

The nick-directed DNA repair efficiency of a set of M13mp18-derived heteroduplexes containing 8-, 12-, 16-, 22-, 27-, 45-, and 429-nucleotide loops was determined by in vitro assay. Unpaired nucleotides of each heteroduplex reside within overlapping recognition sites for two restriction endonucleases, permitting independent evaluation of repair occurring on either DNA strand. Our results show that a strand break located either 3' or 5' to the loop is sufficient to direct heterology repair to the nicked strand in Escherichia coli extracts. Strand-specific repair by this system requires Mg2+ and the four dNTPs and is equally efficient on insertions and deletions. This activity is distinct from the MutHLS mismatch repair pathway. Strand specificity and repair efficiency are largely independent of the GATC methylation state of the DNA and presence of the products of mismatch repair genes mutH, mutL, and mutS. This study provides evidence for a loop repair pathway in E. coli that is distinct from conventional mismatch repair.

Genome Analysis of an Inducible Prophage and Prophage Remnants Integrated in the Streptococcus pyogenes Strain SF370

The mitomycin C inducible prophage SF370.1 from the highly pathogenic M1 serotype Streptococcus pyogenes isolate SF370 showed a 41-kb-long genome whose genetic organization resembled that of SF11-like pac-site Siphoviridae. Its closest relative was prophage NIH1.1 from an M3 serotype S. pyogenes strain, followed by S. pneumoniae phage MM1 and Lactobacillus phage phig1e, Listeria phage A118, and Bacillus phage SPP1 in a gradient of relatedness. Sequence similarity with the previously described prophages SF370.2 and SF370.3 from the same polylysogenic SF370 strain were mainly limited to the tail fiber genes. As in these two other prophages, SF370.1 encoded likely lysogenic conversion genes between the phage lysin and the right attachment site. The genes encoded the pyrogenic exotoxin C of S. pyogenes and a protein sharing sequence similarity with both DNases and mitogenic factors. The screening of the SF370 genome revealed further prophage-like elements. A 13-kb-long phage remnant SF370.4 encoded lysogeny and DNA replication genes. A closely related prophage remnant was identified in S. pyogenes strain Manfredo at a corresponding genome position. The two prophages differed by internal indels and gene replacements. Four phage-like integrases were detected; three were still accompanied by likely repressor genes. All prophage elements were integrated into coding sequences. The phage sequences complemented the coding sequences in all cases. The DNA repair genes mutL and mutS were separated by the prophage remnant SF370.4; prophage SF370.1 and S. pneumoniae phage MM1 integrated into homologous chromosomal locations. The prophage sequences were interpreted with a hypothesis that predicts elements of cooperation and an arms race between phage and host genomes.

The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants.

We have recently described the presence of a high proportion of Pseudomonas aeruginosa isolates (20%) with an increased mutation frequency (mutators) in the lungs of cystic fibrosis (CF) patients. In four out of 11 independent P. aeruginosa strains, the high mutation frequency was found to be complemented with the wild-type mutS gene from P. aeruginosa PAO1. Here, we report the cloning and sequencing of two additional P. aeruginosa mismatch repair genes and the characterization, by complementation of deficient strains, of these two putative P. aeruginosa mismatch repair genes (mutL and uvrD). We also describe the alterations in the mutS, mutL and uvrD genes responsible for the mutator phenotype of hypermutable P. aeruginosa strains isolated from CF patients. Seven out of the 11 mutator strains were found to be defective in the MMR system (four mutS, two mutL and one uvrD). In four cases (three mutS and one mutL), the genes contained frameshift mutations. The fourth mutS strain showed a 3.3 kb insertion after the 10th nucleotide of the mutS gene, and a 54 nucleotide deletion between two eight nucleotide direct repeats. This deletion, involving domain II of MutS, was found to be the main one responsible for mutS inactivation. The second mutL strain presented a K310M mutation, equivalent to K307 in Escherichia coli MutL, a residue known to be essential for its ATPase activity. Finally, the uvrD strain had three amino acid substitutions within the conserved ATP binding site of the deduced UvrD polypeptide, showing defective mismatch repair activity. Interestingly, cells carrying this mutant allele exhibited a fully active UvrABC-mediated excision repair. The results shown here indicate that the putative P. aeruginosa mutS, mutL and uvrD genes are mutator genes and that their alteration results in a mutator phenotype.

Haemophilus influenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli.

MutH, MutL and MutS are essential components of the mismatch repair system in Escherichia coli. Whereas mutS and mutL genes are found in most organisms, the mutH gene is limited to some proteobacteria. We show here that the cloned genes of MutH from Vibrio cholerae and Haemophilus influenzae are able to fully complement a mutH defect in E. coli. Moreover, the purified proteins were shown to be dam methylation sensitive endonucleases, which can be activated by the E. coli MutL protein. These results allow to narrow down regions that are important for the interaction of MutH with MutL.

Haemophilus influenzae and Vibrio cholerae genes for mutH are able to fully complement a mutH defect in Escherichia coli

MutH, MutL and MutS are essential components of the mismatch repair system in Escherichia coli. Whereas mutS and mutL genes are found in most organisms, the mutH gene is limited to some proteobacteria. We show here that the cloned genes of MutH from Vibrio cholerae and Haemophilus influenzae are able to fully complement a mutH defect in E. coli. Moreover, the purified proteins were shown to be dam methylation sensitive endonucleases, which can be activated by the E. coli MutL protein. These results allow to narrow down regions that are important for the interaction of MutH with MutL.

Isolation and characterization of AtMLH1, a MutL homologue from Arabidopsis thaliana

DNA mismatch repair systems play an essential role in the maintenance of genetic information in living organisms and are also implicated in genetic recombination and genome stability. Using degenerate primers, we have cloned the first plant homologue of the E. coli MutL gene, which we have called AtMLH1 for Arabidopsis thaliana MutL-homologue 1. AtMLH1 is present as a single-copy gene in the Arabidopsis genome and is located on the top arm of chromosome 4. Sequence analysis revealed that the product of this gene shows extensive sequence homology with other eukaryotic MLH1 proteins. As mlh1-deficient lines would be useful for studying the biological function of this gene, several populations that had been mutagenized using T-DNA and transposon insertions were screened to identify such mutants. One line that carries a T-DNA insertion in the promoter region of the AtMLH1 gene was isolated. Surprisingly, although the insertion occurred only approximately 80 bp upstream of the putative transcription start site, Northern analyses revealed very low but similar amounts of AtMLH1 transcript in both the wild type and the T-DNA insertion lines. RT-PCR analyses suggest, however, that transcription is initiated further upstream in the insertion line and that the T-DNA may supply this novel initiation site. Finally, no increase in microsatellite instability - a phenotype often associated with mutations in mismatch repair genes - was observed in plants homozygous for this insertion.

Epigenetic phenotypes distinguish microsatellite-stable and -unstable colorectal cancers.

Aberrant DNA methylation is a common phenomenon in human cancer, but its patterns, causes, and consequences are poorly defined. Promoter methylation of the DNA mismatch repair gene MutL homologue (MLH1) has been implicated in the subset of colorectal cancers that shows microsatellite instability (MSI). The present analysis of four MspI/HpaII sites at the MLH1 promoter region in a series of 89 sporadic colorectal cancers revealed two main methylation patterns that closely correlated with the MSI status of the tumors. These sites were hypermethylated in tumor tissue relative to normal mucosa in most MSI(+) cases (31/51, 61%). By contrast, in the majority of MSI(-) cases (20/38, 53%) the same sites showed methylation in normal mucosa and hypomethylation in tumor tissue. Hypermethylation displayed a direct correlation with increasing age and proximal location in the bowel and was accompanied by immunohistochemically documented loss of MLH1 protein both in tumors and in normal tissue. Similar patterns of methylation were observed in the promoter region of the calcitonin gene that does not have a known functional role in tumorigenesis. We propose a model of carcinogenesis where different epigenetic phenotypes distinguish the colonic mucosa in individuals who develop MSI(+) and MSI(-) tumors. These phenotypes may underlie the different developmental pathways that are known to occur in these tumors.

Inversion/dimerization of plasmids mediated by inverted repeats

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8%) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

The Escherichia coli MutL protein stimulates binding of Vsr and MutS to heteroduplex DNA.

Vsr DNA mismatch endonuclease is the key enzyme of very short patch (VSP) DNA mismatch repair and nicks the T-containing strand at the site of a T-G mismatch in a sequence-dependent manner. MutS is part of the mutHLS repair system and binds to diverse mismatches in DNA. The function of the mutL gene product is currently unclear but mutations in the gene abolish mutHLS -dependent repair. The absence of MutL severely reduces VSP repair but does not abolish it. Purified MutL appears to act catalytically to bind Vsr to its substrate; one-hundredth of an equivalent of MutL is sufficient to bring about a significant effect. MutL enhances binding of MutS to its substrate 6-fold but does so in a stoichiometric manner. Mutational studies indicate that the MutL interaction region lies within the N-terminal 330 amino acids and that the MutL multimerization region is at the C-terminal end. MutL mutant monomeric forms can stimulate MutS binding.

Analysis of the Mismatch and Insertion/Deletion Binding Properties ofThermus thermophilus,HB8, MutS

The methyl-directed long patch repair pathway inEsherichia coliis involved in increasing the fidelity of replication specific repair of DNA polymerase incorporation errors. This pathway is mediated by three gene products, MutS, MutL, and MutH, which 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). A MutS homologue has recently been identified in the extremely thermophilic bacterium,Thermus thermophilus.Here we describe analysis of the binding properties of this protein, which has indicated it can identify all specific base mismatches as well as one, two, and three base pair insertion/deletion mutations. We therefore believe this protein may be generally useful for applications involving mismatch detection.

Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis.

The Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150 degrees on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-lacZ transcriptional fusion. An increase in expression was observed during late exponential growth.

Dominant negative mutator mutations in the mutL gene of Escherichia coli.

The mutL gene product is part of the dam-directed mismatch repair system of Escherichia coli but has no known enzymatic function. It forms a complex on heteroduplex DNA with the mismatch recognition MutS protein and with MutH, which has latent endonuclease activity. An N-terminal hexahistidine-tagged MutL was constructed which was active in vivo. As a first stop to determine the functional domains of MutL, we have isolated 72 hydroxylamine-induced plasmid-borne mutations which impart a dominant-negative phenotype to the wild-type strain for increased spontaneous mutagenesis. None of the mutations complement a mutL deletion mutant, indicating that the mutant proteins by themselves are inactive. All the dominant mutations but one could be complemented by the wild-type mutL at about the same gene dosage. DNA sequencing indicated that the mutations affected 22 amino acid residues located between positions 16 and 549 of the 615 amino acid protein. In the N-terminal half of the protein, 12 out of 15 amino acid replacements occur at positions conserved in various eukaryotic MutL homologs. All but one of the sequence changes affecting the C-terminal end of the protein are nonsense mutations.

Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli.

To improve our understanding of the mechanism that couples nucleotide-excision repair to transcription in expressed genes, we have examined the effects of mutations in several different DNA repair genes on the removal of cyclobutane pyrimidine dimers from the individual strands of the induced lactose operon in UV-irradiated Escherichia coli. As expected, we found little repair in either strand of the lactose operon in strains with mutations in established nucleotide excision-repair genes (uvrA, uvrB, uvrC, or uvrD). In contrast, we found that mutations in either of two genes required for DNA-mismatch correction (mutS and mutL) selectively abolish rapid repair in the transcribed strand and render the cells moderately sensitive to UV irradiation. Similar results were found in a strain with a mutation in the mfd gene, the product of which has been previously shown to be required for transcription-coupled repair in vitro. Our results demonstrate an association between mismatch-correction and nucleotide-excision repair and implicate components of DNA-mismatch repair in transcription-coupled repair. In addition, they may have important consequences for human disease and may enhance our understanding of the etiology of certain cancers which have been associated with defects in mismatch correction.

Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.

Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.

Hereditary Nonpolyposis Colorectal Cancer: the Syndrome, the Genes, and Historical Perspectives

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the occurrence within a family of multiple cases of colorectal cancer in the absence of gastrointestinal polyposis. The prevalence of this syndrome is not yet clear, but it may account for 1%-5% of all colorectal cancers. Prior to the identification of the genetic basis of this syndrome, the disease was recognized by the familial aggregation of colorectal cancers that had an early age of onset, an excess of proximally located, and often multiple, primary tumors, and an excess occurrence of cancers in certain other organs. The recent description of an abnormality called "microsatellite instability," present in almost all cancers from HNPCC patients and in about 12%-15% of sporadic cases, led to a series of discoveries that linked this type of genomic instability to a defect in the DNA mismatch repair (MMR) system. Independent investigators have identified four HNPCC genes: hMSH2 (a homologue of the prokaryotic DNA MMR gene MutS) and hMLH1, hPMS1, and hPMS2 (all homologues of the prokaryotic DNA MMR gene MutL). Mutations in each of the four genes have been found in the germline cells of HNPCC families. A major target for research in this area is the development of clinically practical screening tests for the genetic carrier state of HNPCC.

A hPMS2 Mutant Cell Line Is Defective in Strand-specific Mismatch Repair

Human cells contain several homologs of the bacterial mutL gene required for mismatch repair, including a gene on chromosome 7 designated hPMS2. We have identified an endometrial carcinoma cell line, HEC-1-A, that has a C-->T mutation in hPMS2 that generates a nonsense codon and yields a protein truncated at the C terminus. No wild-type gene or gene product was detected. The missing amino acids in hPMS2 are highly conserved among PMS homologs, suggesting that they may be critical for function. In support of this, extracts of HEC-1-A cells are defective in repairing a variety of mismatched substrates. Moreover, di-, tri-, and tetranucleotide repeated sequences are highly unstable in single cell clones of HEC-1-A cells, and HEC-1-A cells are resistant to killing by N-methyl-N'-nitro-N-nitrosoguanidine. The results provide strong experimental support for the involvement of the hPMS2 gene product in mismatch repair in human cells and support the concept that a defective hPMS2 gene may lead to predisposition to certain forms of cancer.

Cloning, Characterization and Chromosomal Assignment of the Human Genes Homologous to Yeast PMS1, a Member of Mismatch Repair Genes

Mutations in genes associated with the DNA mismatch repair system were considered to play important roles in predisposition to cancer, since hMSH2 and hMLH1, human homologues of yeast MSH2 and MLH1 as well as bacterial mutS and mutL genes, were found to be involved in hereditary nonpolyposis colorectal cancer (HNPCC). In addition, yeast PMS1 that is homologous to bacterial mutL and hexB, has also been proven to be related to the DNA mismatch repair system. As the first step to understand whether human homologue of the yeast PMS1 gene is associated with genetic predisposition to cancer, we have isolated and analyzed human counterpart of yeast PMS1 genes. DNA sequencing analyses indicated that human PMS genes constituted a multiple gene family and that some of the family members have been mapped to chromosomal bands 7q11.23 and 7q22 by fluorescent in situ hybridization.

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.

Transcriptional patterns of the mutL-miaA superoperon of Escherichia coli K-12 suggest a model for posttranscriptional regulation

The complex amiB-mutL-miaA-hfq-hflX-hflK-hflC superoperon of E coli contains important genes for several fundamental cellular processes, including cell-wall hydrolysis ( amiB), DNA repair ( mutL), tRNA modification ( miaA) and proteolysis ( hflX-hflK-hflC). We report here the transcriptional pattern and possible posttranscriptional regulation of mutL, miaA and hfq genes of this superoperon. RNase protection analysis of mRNA transcribed from the bacterial chromosome demonstrated that there is co-transcription of mutl and miaA. In addition, two internal promoters, P miaA and P1 hfq were identified and mapped to 201 and 837 nucleotides upstream from the respective translation start sites. P miaA contains poor matches to the −10 and −35 regions of the sigma-70 RNA polymerase concensus sequences, but it contains multiple potential Fis-binding sites and an upstream AT-rich region with poly(A) sequences. The basic arrangement of Fis-binding sites followed by an AT rich region is shared with promoters for rRNA operons and some of the tRNA and tRNA modification genes. As part of an initial study of mutL and miaA regulation, we measured transcript amounts in isogenic rne, rnc and rne rnc double mutants which are deficient in RNase E, RNase III or both. The amounts of steady state level mutL-miaA cotranscript, P miaA transcript and Pl hfq transcript increased eight-, nine- and three-fold respectively in an rne3071 mutant when compared to the rne + parent. In contrast, amounts of the three transcripts were the same in an rnc105 mutant and its rnc + parent. These results indicate that mutL, miaA, and hfq expression could be regulated by multiple mechanisms, including degree of cotranscription from upstream genes, modulation of internal promoter strength, and by RNase E activity. A model is presented for RNase E-mediated posttranscriptional regulation that may coordinated mutL expression with replication and miaA with tRNA amounts under different growth conditions, especially during nutrient upshifts.

Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene.

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.