Cloning and characterization of mutL and mutS genes of Vibrio cholerae: nucleotide sequence of the mutL gene.

The mutL and mutS genes of Vibrio cholerae have been identified using interspecific complementation of Escherichia coli mutL and mutS mutants with plasmids containing the gene bank of V. cholerae. The recombinant plasmid pJT470, containing a 4.7 kb fragment of V. cholerae DNA codes for a protein of molecular weight 92,000. The product of this gene reduces the spontaneous mutation frequency of the E. coli mutS mutant. The plasmid, designated pJT250, containing a 2.5 kb DNA fragment of V. cholerae and coding for a protein of molecular weight 62,000, complements the mutL gene function of E. coli mutL mutants. These gene products are involved in the repair of mismatches in DNA. The complete nucleotide sequence of mutL gene of V. cholerae has been determined.     

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.     

Identification and characterization of the mutL and mutS gene products of Salmonella typhimurium LT2.

The gene products of the mutL and mutS loci play essential roles in the dam-directed mismatch repair in both Salmonella typhimurium LT2 and Escherichia coli K-12. Mutations in these genes result in a spontaneous mutator phenotype. We have cloned the mutL and mutS genes from S. typhimurium by generating mutL- and mutS-specific probes from an S. typhimurium mutL::Tn10 and an mutS::Tn10 strain and using these to screen an S. typhimurium library. Both the mutL and mutS genes from S. typhimurium were able to complement E. coli mutL and mutS strains, respectively. By a combination of Tn1000 insertion mutagenesis and the maxicell technique, the products of the mutL and mutS genes were shown to have molecular weights of 70,000 and 98,000 respectively. A phi (mutL'-lacZ+) gene fusion was constructed; no change in the expression of the fusion could be detected by treatment with DNA-damaging agents. In crude extracts, the MutS protein binds single-stranded DNA, but not double-stranded DNA, with high affinity.     

The specificity of base-pair substitution induced by the mutL and mutS mutators in E. coli

The Escherichia coli mutator alleles, mutL and mutS, produced transversion as well as transition base-pair substitutions with the trpA reversion system. Transversions, however, were generally mutator-induced at a lower level than transitions and the specific type of transversion and its nucleotide position appeared to strongly affect its level of enhancement. These results are interpreted to mean that mutL- and mutS-dependent mismatch correction is generally more effective at correcting transition mispairings than transversion mispairings. Correction of transversion mispairings is probably dependent upon site of occurrence and type of mismatch.     

Mutagenesis, by methylating and ethylating agents, in mutH, mutL, mutS, and uvrD mutants of Salmonella typhimurium LT2.

Salmonella typhimurium LT2 mutH, mutL, mutS, and uvrD mutants were especially sensitive to mutagenesis by both the recA+-dependent mutagen methyl methane sulfonate and the recA+-independent mutagen ethyl methane sulfonate, but not to mutagenesis by agents such as 4-nitroquinoline-1-oxide and UV irradiation. Similarly, these mutator strains were very sensitive to mutagenesis by the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea. The increased susceptibility to mutagenesis by small alkylating agents due to mutH, mutL, mutS, and uvrD mutations was not accompanied by an increased sensitivity to killing by these agents. Various models are discussed in an effort to explain why strains thought to be deficient in methyl-instructed mismatch repair are sensitive to mutagenesis by methylating and ethylating agents.     

5-methylcytosine at HpaII sites in p53 is not hypermutable after UVC irradiation

Using a yeast based p53 functional assay we previously demonstrated that the UVC-induced p53 mutation spectrum appears to be indistinguishable from the one observed in Non Melanoma Skin Cancer (NMSC). However, position 742 (codon 248, CpG site) represented the major hot spot in NMSC but was not found mutated in the yeast system. In order to determine whether UVC-induced mutagenic events may be facilitated at methylated cytosine (5mC), a yeast expression vector harbouring a human wild-type p53 cDNA (pLS76) was methylated in vitro by HpaII methylase. Methylation induced 98% protection to HpaII endonuclease. Unmethylated and methylated pLS76 vectors were then UVC irradiated (lambda(max): 254 nm) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results revealed that: (i) 5mC at HpaII sites did not cause any difference in the UVC-induced survival and/or mutagenicity; (ii) none of the 20 mutants derived from methylated pLS76 showed p53 mutations targeted at HpaII sites; (iii) the UVC-induced p53 mutation spectra derived from methylated and unmethylated pLS76 were indistinguishable not only when classes of mutations and hot spots were concerned, but also when compared through a rigorous statistical test to estimate their relatedness (P = 0.85); (iv) the presence of 5mC did not increase the formation of photo-lesions at codon 248, as determined by using a stop polymerase assay. Although based on a limited number of mutants, these results suggest that the mere presence of 5mC at position 742 does not cause a dramatic increase of its mutability after UVC irradiation. We propose that position 742 is a hot spot in NMSC either because of mutagenic events at 5mC caused by other UV components of solarlight and/or because not all the NMSC are directly correlated with UV mutagenesis but may have a "spontaneous" origin.     

Bacterial DNA repair genes and their eukaryotic homologues: 5. The role of recombination in DNA repair and genome stability

Recombinational repair is a well conserved DNA repair mechanism present in all living organisms. Repair by homologous recombination is generally accurate as it uses undamaged homologous DNA molecule as a repair template. In Escherichia coli homologous recombination repairs both the double-strand breaks and single-strand gaps in DNA. DNA double-strand breaks (DSB) can be induced upon exposure to exogenous sources such as ionizing radiation or endogenous DNA-damaging agents including reactive oxygen species (ROS) as well as during natural biological processes like conjugation. However, the bulk of double strand breaks are formed during replication fork collapse encountering an unrepaired single strand gap in DNA. Under such circumstances DNA replication on the damaged template can be resumed only if supported by homologous recombination. This functional cooperation of homologous recombination with replication machinery enables successful completion of genome duplication and faithful transmission of genetic material to a daughter cell. In eukaryotes, homologous recombination is also involved in essential biological processes such as preservation of genome integrity, DNA damage checkpoint activation, DNA damage repair, DNA replication, mating type switching, transposition, immune system development and meiosis. When unregulated, recombination can lead to genome instability and carcinogenesis.     

DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine.

While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C). Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy. Eight of the bacterial DNAs contained m4C. Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile. The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs. Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns. Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes. Instead, some of them have deamination-resistant m4C residues.     

Distribution of 5-methylcytosine residues in 5S rRNA genes in Arabidopsis thaliana and Secale cereale

Bisulfite genomic sequencing was used to localise 5-methylcytosine residues (mC) in 5S rRNA genes of Arabidopsis thaliana and Secale cereale. The maps of mC distribution were compared with the previously published map of the corresponding region in Nicotiana tabacum. In all three species, the level of methylation of 5S rRNA genes was generally higher than the average for the entire genome. The ratio of 5S rDNA methylation to average overall methylation was 44%/30-33% for N. tabacum, 27%/4-6% for A. thaliana and 24%/20-22% for S. cereale. With the exception of one clone from S. cereale, no methylation-free 5S rDNA was detected. The level of methylation at different sequence motifs in 5S rDNA was calculated for N. tabacum/A. thaliana/ S. cereale, and this analysis yielded the following values (expressed as a percentage of total C): mCG 90%/78%/85%, mCWG 89%/41%/53%, mCmCG 72%/32%/16%, mCCG 4%/2%/0%, mCHH 15%/6%/1%, where W=A or T, and H=A or C or T. Non-symmetrical methylation was almost negligible in the large genome of S. cereale but relatively frequent in N. tabacum and A. thaliana, suggesting that the strict correlation between genome size and cytosine methylation might be violated for this type of methylation. Among non-symmetrical motifs the mCWA triplets were significantly over-represented in Arabidopsis, while in tobacco this preference was not as pronounced. The differences in methylation levels in different sequence contexts might be of phylogenetic significance, but further species in related and different taxa need to be studied before firm conclusions can be drawn.     

Detection of 5-methylcytosine in DNA sequences.

Col E1 DNA has methylated cytosine in the sequence 5'-CC*(A/T)GG-3' and methylated adenine in the sequence 5'-GA*TC-3' at the positions indicated by asterisks(*). When the Maxam-Gilbert DNA sequencing method is applied to this DNA, the methylated cytosine (5-methylcytosine) is found to be less reactive to hydrazine than are cytosine and thymine, so that a band corresponding to that base does not appear in the pyrimidine cleavage patterns. The existence of the methylated cytosine can be confirmed by analyzing the complementary strand or unmethylated DNA. In contrast, the methylated adenine (probably N6-methyladenine) cannot be distinguished from adenine with standard conditions for cleavage at adenine.     

Involvement of 5-methylcytosine in sunlight-induced mutagenesis.

In human skin cancers, more than 30 % of all mutations in the p53 gene are transitions at dipyrimidines within the sequence context CpG, i.e. 5'-TCG and 5'-CCG, found at several mutational hotspots. Since CpGs are methylated along the p53 gene, these mutations may be derived from solar UV-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. In Xorder to define the contribution of 5-methylcytosine to sunlight-induced mutations, we have used mouse fibroblasts containing the CpG-methylated lacI transgene as a mutational target. We sequenced 182 UVC (254 nm UV)-induced mutations and 170 mutations induced by a solar UV simulator, along with 75 mutations in untreated cells. Only a few of the mutations in untreated cells were transitions at dipyrimidines, but more than 95% of the UVC and solar irradiation-induced mutations were targeted to dipyrimidine sites, the majority being transitions. After UVC irradiation, 6% of the base substitutions were at dipyrimidines containing 5-methylcytosine and only 2.2% of all mutations were transitions within this sequence context. However, 24% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of them were transitions. Two sunlight-induced mutational hotspots at methylated CpGs correlated with sequences that form the highest levels of cyclobutane pyrimidine dimers after irradiation with sunlight but not with UVC. The data indicate that dipyrimidines that contain 5-methylcytosine are preferential targets for sunlight-induced mutagenesis in cultured mammalian cells, thus explaining the large proportion of p53 mutations at such sites in skin tumors in vivo.     

Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing

Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase.     

Synthesis and physical characterization of DNA fragments containing N4-methylcytosine and 5-methylcytosine.

The synthesis of N4-methyl-2'-deoxycytidine and its fully protected mononucleotide, suitable for the oligonucleotide synthesis by phosphotriester method is described. A set of octanucleotides - d(CGCGCGCG), d(CG5mCGCGCG), d(CG4mCGCGCG) and dodecanucleotides - d(GGACCCGGGTCC), d(GGA5mCCCGGGTCC), d(GGA4mCCCGGGTCC) has been synthesized in a solution. Physical characterization of the oligonucleotide duplexes by means of UV and CD spectrometry provides the evidence that 4mC similarly to 5mC favours the B--greater than Z transition, although both of these methylated cytosines inhibit the B--greater than A conformational change. N4-Methylcytosine in contrast to 5-methylcytosine reduces the DNA double helix thermal stability.     

Identifying 5-methylcytosine and related modifications in DNA genomes.

Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the principles and applications of each method, quantitatively evaluated their specificity,resolution and sensitivity, identified potential artifacts and suggested solutions, and discussed a paradox in the distribution of m5C in mammalian genomes that illustrates how methodological limitations can affect interpretation of data. Hopefully, the information and analysis provided here will guide new investigators entering this exciting field.     

Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing

Genomic sequencing was used to localise 5-methylcytosine residues in individual DNA strands of 5S rRNA genes in tobacco. The density of methylation along the sequence was high in both strands, exceeding the average methylation density of the tobacco genome. Besides methylation of CG and CNG sequences, considerable amounts of mC were found in non-symmetrical sites. Among 69 sequenced clones obtained from leaf DNA we did not detect any non-methylated clone, and Southern blot hybridisation analysis also failed to suggest the presence of methylation-free 5S rDNA units in the tobacco genome. Differences were observed among methylation patterns of individual sequenced clones. This heterogeneity reflects either heterogeneity among individual members of 5S rRNA gene cluster or differences among individual cells. Methylation of CNG and non-symmetrical sites can be efficiently reduced by treatment with dihydroxypropyladenine, an inhibitor of S-adenosylhomocysteine hydrolase. Received: 28 January 1998 / Accepted: 29 April 1998