A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Establishment of a novel sensitive method for detecting methylation modification on DNA of escherichia coli cell

This study focused on finding a novel sensitive method to determine the methylation modification at DNA dam (GATC) sites in Escherichia coli. A new plasmid which contained three GATC sites recognized by restriction enzyme BclI and one GAATTC site recognized by EcoRI was transformed into E. coli stains AB1157(dam ⁺) and GM2929(dam ⁻) respectively. Then the plasmid DNA was digested by restriction enzyme BclI(T*GATCA), which was sensitive to methylation. The results showed that the plasmid derived from AB1157 was not digested while that from GM2929 was, for the methylation level of the former was high while the latter was low. So by detecting the methylation of plasmid transferred into the strain, we could determine whether methylaion existed at DNA dam (GATC) site in E. coli. This method was effective and rapid; moreover, the digested fragments were not dispersive. It also made a basis for the detection of whether methylation occurred in mode beings by low-energy ion beam. The article is published in the original.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Stationary-state mutagenesis inEscherichia coli: A model

Stationary-phase mutagenesis in nondividingE. coli cells exposed to a nonlethal stress was, a few years ago, claimed to be a likely case of a Lamarckian mechanism capable of producing exclusively useful mutations in a directed manner. After a heated debate over the last decade it now appears to involve a Darwinian mechanism that generates a transient state of hypermutagenesis, operating on a large number of sites spread over the entire genome, at least in a proportion of the resting cells. Most of the studies that clarified this position were on the reversion of a frameshift mutation present in alacI-lacZ fusion inE. coli strain FC40. Several groups have extensively examined both the sequence changes associated with these reversions and the underlying genetic requirements. On the basis of our studies on the genomic sequence analysis, we recently proposed a model to explain the specific changes associated with the reversion hotspots. Here we propose a more detailed version of this model that also takes into account the observed genetic requirements of stationary-state mutagenesis. Briefly, G:T/U mismatches produced at methylatable cytosines are preferentially repaired in nondividing cells by the very short patch mismatch repair (VSPMR) mechanism which is itself mutagenic and can produce mutations in very short stretches located in the immediate vicinity of these cytosine methylation sites. This mechanism requires a homologous or homeologous strand invasion step and an error-prone DNA synthesis step and is dependent on RecA, RecBCD and a DNA polymerase. The process is initiated near sequences recognized by Dcm and Vsr enzymes and further stimulated if these sequences are a part of CHI or CHI-like sequences, but a double-strand-break-dependent recombination mediated by the RecBCD pathways proposed by others seems to be nonessential. The strand transfer step is proposed to depend on RecA, RuvA, RuvB and RuvC and is opposed by RecG and MutS. The model also gives interesting insights into the evolution of theE. coli genome.     

Selection againstdam methylation sites in the genomes of DNA of enterobacteriophages

Summary Postreplicative methylation of adenine inEscherichia coli DNA to produce G^6m ATC (where^6mA is 6-methyladenine) has been associated with preferential daughter-strand repair and possibly regulation of replication. An analysis was undertaken to determine if these, or other, as yet unknown roles of GATC, have had an effect on the frequency of GATC inE. coli or bacteriophage DNA. It was first ascertained that the most accurate predictions of GATC frequency were based on the observed frequencies of GAT and ATC, which would be expected since these predictors take into account preferences in codon usage. The predicted frequencies were compared with observed GATC frequencies in all available bacterial and phage nucleotide sequences. The frequency of GATC was close to the predicted frequency in most genes ofE. coli and its RNA bacteriophages and in the genes of nonenteric bacteria and their bacteriophages. However, for DNA enterobacteriophages the observed frequency of DNA enterobacteriophages the observed frequency of GATC was generally significantly lower than predicted when assessed by the chi square test. No elevation in the rate of mutation of^6mA in GATC relative to other bases was found when pairs of DNA sequences from closely related phages or pairs of homologous genes from enterobacteria were compared, nor was any preferred pathway for mutation of^6mA evident in theE. coli DNA bacteriophages. This situation contrasts with that of 5-methylcytosine, which is hypermutable, with a preferred pathway to thymine. Thus, the low level of GATC in enterobacteriophages is probably due not to^6mA hypermutability, but to selection against GATC in order to bypass a GATC-mediated host function.     

DNA methylation in mycobacteria: Absence of methylation at GATC (Dam) and CCA/TGG (Dcm) sequences

Abstract The presence of 6-methyladenine and 5-methylcytosine at Dam (GATC) and Dcm (CCA/TGG) sites in DNA of mycobacterial species was investigated using isoschizomer restriction enzymes. In all species examined, Dam and Dcm recognition sequences were not methylated indicating the absence of these methyltransferases. On the other hand, high performance liquid chromatographic analysis of genomic DNA from Mycobacterium smegmatis and Mycobacterium tuberculosis showed significant levels of 6-methyladenine and 5-methylcytosine suggesting the presence of DNA methyltransferases other than Dam and Dcm. Occurrence of methylation was also established by a sensitive genetic assay.     

The possible involvement of CHI sequences in adaptive mutagenesis: Evidence from sequence analysis

Recently we proposed that sequences in the immediate neighbourhood of cytosine residues whose sequence context permits their methylation by DNA cytosine methyltransferase (Dcm) experience hypermutagenesis in cells exposed to nonlethal stresses. This hypothesis could explain the peculiar spectrum of the late-arising Lac⁺ mutants seen in theE. coli strain FC40. Here we present results of computer analysis which show that Dcm substrate sequences are overrepresented in theE. coli genome. Interestingly, certain noncanonical Dcm sequences are more overrepresented than the canonical one. The most overabundant of these, DCM-III (5’ GCTGG3’), forms the 5’ end of the recombinogenic octamer CHI (5’ GCTGGTGG3’). CHI is even more overrepresented than DCM-III. We propose that the overabundance of the DCM and CHI sequences is due to their ability to enhance adaptive fitness of the host by inducing hypermutagenesis in cells exposed to nonlethal, growth-blocking stresses. The CHI context seems to stimulate the adaptive activity of DCM-III by a mechanism which may not directly involve its recombinogenic activity.     

Mutation probe of gene structure in E. coli: suppressor mutations in the seven-tRNA operon

Summary Cells defective in uracil-DNA glycosylase (ung:: Tn10) were used in two ways to reveal differences in select point mutations (GC to AT transitions) within the seven-tRNA operon of E. coli. The mutations were indicated as de novo or converted glutamine tRNA suppressor mutations in the genes glnU and/or glnV: (1) the kinetics of photoenzymatic monomerization of pyrimidine dimers quantitated by ung-dependent UV mutagenesis indicated more rapid repair of dimers at sites for converted suppressor mutation than of dimers at sites for de novo suppressor mutation, and (2) spontaneous deamination of cytosine was considerably more frequent at sites for converted suppressor mutation than at sites for de novo suppressor mutation. To explain these results we suggest the physical structure of the DNA in vivo is different at different sites in the seven-tRNA operon. The non-transcribed strand including specifically the anticodon region of the site for converted suppressor mutation may frequently be looped out in a single strand so that a T=C dimer is more accessible to DNA photolyase or a free cytosine residue of non-irradiated DNA is in an aqueous environment conducive to deamination. In addition, we analysed the spontaneous de novo suppressor mutation data to determine an estimate for the in vivo rate of cytosine deamination in double strand DNA of 3.2×10¹³/sec.     

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

Summary The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 by away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam ^– mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB

Summary Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes. The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification. The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA. The mutations map in the vicinity of the serB locus. This and other data indicate that the host rglB function is involved in the discrimination against modified DNA.     

Systematic Proteomic Analysis of Protein Methylation in Prokaryotes and Eukaryotes Revealed Distinct Substrate Specificity

The studies of protein methylation mainly focus on lysine and arginine residues due to their diverse roles in essential cellular processes from gene expression to signal transduction. Nevertheless, atypical protein methylation occurring on amino acid residues, such as glutamine and glutamic acid, is largely neglected until recently. In addition, the systematic analysis for the distribution of methylation on different amino acids in various species is still lacking, which hinders our understanding of its functional roles. In this study, we deeply explored the methylated sites in three species Escherichia coli, Saccharomyces cerevisiae, and HeLa cells by employing MS‐based proteomic approach coupled with heavy methyl SILAC method. We identify a total of 234 methylated sites on 187 proteins with high localization confidence, including 94 unreported methylated sites on nine different amino acid residues. KEGG and gene ontology analysis show the pathways enriched with methylated proteins are mainly involved in central metabolism for E. coli and S. cerevisiae, but related to spliceosome for HeLa cells. The analysis of methylation preference on different amino acids is conducted in three species. Protein N‐terminal methylation is dominant in E. coli while methylated lysines and arginines are widely identified in S. cerevisiae and HeLa cells, respectively. To study whether some atypical protein methylation has biological relevance in the pathological process in mammalian cells, we focus on histone methylation in diet‐induced obese (DIO) mouse. Two glutamate methylation sites showed statistical significance in DIO mice compared with chow‐fed mice, suggesting their potential roles in diabetes and obesity. Together, these findings expanded the methylome database from microbes to mammals, which will benefit our further appreciation for the protein methylation as well as its possible functions on disease.     

综述: 表观遗传学研究的模式生物——粗糙脉孢菌

丝状真菌粗糙脉孢菌是一种作为遗传学研究的经典模式生物．通过对粗糙脉孢菌5S rRNA基因的组成和在染色体上分布的研究,揭示了丝状真菌中存在的一种基因组防御机制——重复序列诱导的DNA点突变(RIP)．通过对发生突变的5S rRNA假基因的研究还发现,粗糙脉孢菌中存在一种重要的表观遗传修饰——DNA甲基化,随后的深入研究使粗糙脉孢菌成为解析DNA甲基化机制的最重要模式生物之一．粗糙脉孢菌基因转化操作引起的营养生长阶段同源基因的沉默(quelling)是由RNAi途径调控的,同时该途径也是调控减数分裂过程中非配对DNA诱发的基因沉默(meiotic silencing)的关键．由于粗糙脉孢菌基因组简单,且存在与高等真核生物相同的DNA甲基化和多种组蛋白的修饰,使其成为今后深入研究组蛋白修饰与染色质重塑等表观遗传现象参与基因表达调控和基因组稳定性维持的重要模式生物之一．     

Inhibition of cytosine methylation allows efficient cloning of T-DNA tagged plant DNA ofArabidopsis thaliana by plasmid rescue

Summary Plasmid rescue can provide an efficient way of cloning T-DNA-tagged genomic DNA of plants. However, rescue has often been hampered by extensive rearrangements in the cloned DNA. We have demonstrated using a transgenic line ofArabidopsis thaliana that the plant DNA flanking the T-DNA tag was heavily cytosine methylated. This methylation could be completely inhibited by growing the plants in the presence of azacytidine. Rescue of the T-DNA tag together with the flanking plant genomic DNA sequences from nontreated control plants into an modified cytosine restriction (mcr) proficient strain ofEscherichia coli resulted in rearrangements of the majority of the rescued plasmids. These rearrangements could be avoided if the methylation was inhibited in the transgenic plants by azacytidine treatment or by cloning into anmcr-deficient strain ofE. coli. The results indicate that cytosine methylation of the DNA in the transgenic plants is the main cause of the DNA rearrangements observed during plasmid rescue and suggest efficient strategies to eliminate such artifacts.     

Compositional heterogeneity of the Escherichia coli genome: A role for VSP repair?

E. coli genes that contain a high frequency of the tetranucleotide CTAG are also rich in the tetramers CTTG, CCTA, CCAA, TTGG, TAGG, and CAAG (group-I tetramers). Conversely, E. coli genes lacking CTAG are rich in the tetranucleotides CCTG, CCAG, CTGG, and CAGG (group-II tetramers). These two gene samples differ also in codon usage, amino acid composition, frequency of Dcm sites, and contrast vocabularies. Group-I tetramers have in common that they are depleted by very-short-patch repair (VSP), while group-II tetramers are favored by VSP activity. The VSP system repairs G:T mismatches to G:C, thereby increasing the overall G+C content of the genome; for this reason the CTAG-rich sample has a lower G+C content than the CTAG-poor sample. This compositional heterogeneity can be tentatively explained by a low level of VSP activity on the CTAG-rich sample. A negative correlation is found between the frequency of group-I tetramers and the level of gene expression, as measured by the Codon Adaptation Index (CAI). A possible link between the rate of VSP activity and the level of gene expression is considered.Correspondence to: A. Marine     

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5''-CpG-3'' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.¹ and Clark et al.², methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.