A gene required for very short patch repair in Escherichia coli is adjacent to the DNA cytosine methylase gene.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mismatches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previously we have shown that a plasmid containing an 11-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. In the process, we have constructed plasmids that complement the chromosomal mutation for methylation, but not for repair, and vice versa. The genes responsible for these phenotypes have been identified by DNA sequence analysis. The gene essential for cytosine methylation, dcm, is predicted to code for a 473-amino-acid protein and is not required for VSP repair. It is similar to other DNA cytosine methylases and shares extensive sequence similarity with its isoschizomer, EcoRII methylase. The segment of DNA essential for VSP repair contains a gene that should code for a 156-amino-acid protein. This gene, named vsr, is not essential for DNA methylation. Remarkably, the 5' end of this gene appears to overlap the 3' end of dcm. The two genes appear to be transcribed from a common promoter but are in different translational registers. This gene arrangement may assure that Vsr is produced along with Dcm and may minimize the mutagenic effects of cytosine methylation.     

Crystal structure of the Escherichia coli dcm very-short-patch DNA repair endonuclease bound to its reaction product-site in a DNA superhelix

Very-short-patch repair (Vsr) enzymes occur in a variety of bacteria, where they initiate nucleotide excision repair of G:T mismatches arising by deamination of 5-methyl-cytosines in specific regulatory sequences. We have now determined the structure of the archetypal dcm-Vsr endonuclease from Escherichia coli bound to the cleaved authentic hemi-deaminated/hemi-methylated dcm sequence 5′-C-OH-3′ 5′-p-T-p-A-p-G-p-G-3′/3′-G-p-G-p-T-p^Me5C-p-C formed by self-assembly of a 12mer oligonucleotide into a continuous nicked DNA superhelix. The structure reveals the presence of a Hoogsteen base pair within the deaminated recognition sequence and the substantial distortions of the DNA that accompany Vsr binding to product sites.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.     

Structure and expression of the alkB gene of Escherichia coli related to the repair of alkylated DNA

When the alkB gene of Escherichia coli that controls sensitivity of bacteria to methyl methanesulfonate was placed under the control of the lac regulatory region on a multicopy plasmid, the gene product, AlkB protein, was overproduced. By monitoring the band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was purified to near physical homogeneity. An amino-terminal sequence and total amino acid composition of the purified AlkB protein were in accord with the amino acid sequence deduced from the nucleotide sequence of the alkB gene, determined by the phage M13 dideoxy method. It was concluded that the AlkB protein is comprised of 216 amino acids and has a molecular weight of 23,900. The nucleotide sequence analysis also revealed that the ada and alkB genes are adjacent on the E. coli chromosome and that the first initiation codon for AlkB protein overlaps with the termination codon for Ada protein. We constructed hybrid plasmids carrying an alkB'-lacZ' fusion, with or without the ada control region, and investigated expression of the alkB gene in response to the alkylating agent. We obtained evidence that the ada and alkB genes constitute an operon.     

Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12

Summary A mutation in the purB gene of E. coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30°C. Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work. The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair. Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response. DNA isolated from mud cells is not undermethylated relative to DNA from purB ⁺ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues. Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination. Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.This work was supported in part by Public Health Service grants numbers GM15697 and CA32182     

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase

BackgroundControl of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans.MethodsC-5 methyl cytosines (m⁵C) were detected by immuno-blotting with m⁵C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry.ResultsMethylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m⁴C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m⁵C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc.ConclusionDR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m⁵C.General significanceAlthough, dispensable under optimal growth conditions, the presence of m⁵C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.     

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35?years ago, the biological role of 5-methylcytosine in E.?coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.?coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.     

Role of uracil-DNA glycosylase in the repair of deaminated cytosine residues of DNA in Escherichia coli.

Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of Escherichia coli 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. piX174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of E. coli mutant BD10 (ung). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.     

Location of the gene specifying cytosine deaminase in Escherichia coli

Molecular Genetics and Genomics - The gene specifying cytosine deaminase (cod) is shown to be located at approximately 86 minutes on the linkage map of E. coli. The corresponding gene in S....     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.     

Location of gene specifying cytosine deaminase in Escherichia coli

Summary The gene specifying cytosine deaminase (cod) is shown to be located at approximately 86 minutes on the linkage map of E. coli. The corresponding gene in S. typhimurium has been reported to have a different location.     

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.     

Escherichia coli cis- and trans-acting mutations that increaseglyA gene expression

We used anEscherichia coli strain blocked in serine biosynthesis and carrying a partialglyA deletion to isolate strains with altered regulation of theglyA gene. TheglyA deletion results in 25% of the normal serine hydroxymethyltransferase activity. Three classes of mutants with increasedglyA expression were isolated on glycine supplemented plates. One class of mutations increasedglyA expression 10-fold by directly altering the – 35 consensus sequence of theglyA promoter. The two other classes increasedglyA expression about 2- and 6-fold, respectively. The latter two classes of mutations also affected regulation of themetE gene of the folate branch of the methionine pathway, but notmetA in the nonfolate branch of the methionine pathway, or thegcv operon, encoding the glycine cleavage enzyme system. The mutations were mapped to about minute 85.5 on theE. coli chromosome.     

Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli, using gene expression profiles from DNA microarrays

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.     

Escherichia coli K-12 clones that overproduce dam methylase are hypermutable.

A strain of Escherichia coli K-12 that overproduces dam methylase 50-fold was found to be hypermutable, and mutations which resulted in loss of excess methylase activity restored mutation frequencies to wild-type levels. These results are consistent with involvement of this deoxyribonucleic acid methylase in mismatch correction.