Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA. Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites. We have determined that the damh mutation produces a single amino acid change (Pro126 to Ser126) in a region of homology (III) shared by three DNA-adenine methyltransferases; viz, T4 Dam, Escherichia coli Dam, and the DpnII modification enzyme of Streptococcus pneumoniae. We also describe another mutant, damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA. This mutation also alters a single amino acid (Phe127 to Val127). These results implicate homology region III as a domain involved in DNA sequence recognition. The effect of several different amino acids at residue 126 was examined by creating a polypeptide chain terminating codon at that position and comparing the methylation capability of partially purified enzymes produced in the presence of various suppressors. No enzyme activity is detected when phenylalanine, glutamic acid, or histidine is inserted at position 126. However, insertion of alanine, cysteine, or glycine at residue 126 produces enzymatic activity similar to Damh.
Bacteriophages T2 and T4 encode DNA-[N6-adenine] methyltransferases (Dam) which differ from each other by only three amino acids. The canonical recognition sequence for these enzymes in both cytosine and 5-hydroxymethylcytosine-containing DNA is GATC; at a lower efficiency they also recognize some non-canonical sites in sequences derived from GAY (where Y is cytosine or thymine). We found that T4 Dam fails to methylate certain GATA and GATT sequences which are methylated by T2 Dam. This indicates that T2 Dam and T4 Dam do not have identical sequence specificities. We analyzed DNA sequence data files obtained from GenBank, containing about 30% of the T4 genome, to estimate the overall frequency of occurrence of GATC, as well as non-canonical sites derived from GAY. The observed N6methyladenine (m6A) content of T4 DNA, methylated exclusively at GATC (by Escherichia coli Dam), was found to be in good agreement with this estimate. Although GATC is fully methylated in virion DNA, only a small percentage of the non-canonical sequences are methylated.
The DNA of Serratia marcescens has N6-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from S. marcescens Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (dam) coding for the adenine methyltransferase (Dam enzyme) of S. marcescens was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a dam-13::Tn9 mutant of Escherichia coli. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of E. coli. The dam gene is located between flanking genes which are similar to those found to the sides of the E. coli dam gene. The results of complementation studies indicated that like Dam of E. coli and unlike Dam of Vibrio cholerae, the Dam enzyme of S. marcescens plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors.
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.
The T4 dam+ gene has been cloned (S. L. Schlagman and S. Hattman, Gene 22:139-156, 1983) and transferred into an Escherichia coli dam-host. In this host, the T4 Dam DNA methyltransferase methylates mainly, if not exclusively, the sequence 5'-GATC-3'; this sequence specificity is the same as that of the E. coli Dam enzyme. Expression of the cloned T4 dam+ gene suppresses almost all the phenotypic traits associated with E. coli dam mutants, with the exception of hypermutability. In wild-type hosts, 20- to 500-fold overproduction of the E. coli Dam methylase by plasmids containing the cloned E. coli dam+ gene results in a hypermutability phenotype (G.E. Herman and P. Modrich, J. Bacteriol. 145:644-646, 1981; M.G. Marinus, A. Poteete, and J.A. Arraj, Gene 28:123-125, 1984). In contrast, the same high level of T4 Dam methylase activity, produced by plasmids containing the cloned T4 dam+ gene, does not result in hypermutability. To account for these results we propose that the E. coli Dam methylase may be directly involved in the process of methylation-instructed mismatch repair and that the T4 Dam methylase is unable to substitute for the E. coli enzyme.
A gene from the periodontal organism Porphyromonas gingivalis has been identified as encoding a DNA methylase. The gene, referred to as pgiIM, has been sequenced and found to contain a reading frame of 864 basepairs. The putative amino acid sequence of the encoded methylase was 288 amino acids, and shared 47% and 31% homology with the Streptococcus pneumoniae DpnII and E. coli Dam methylases, respectively. The activity and specificity of the pgi methylase (M.PgiI) was confirmed by cloning the gene into a dam- strain of E. coli (JM110) and performing a restriction analysis on the isolated DNA with enzymes whose activities depended upon the methylation state of the DNA. The data indicated that M.PgiI, like DpnII and Dam, methylated the adenine residue within the sequence 5'-GATC-3'.
The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in kcat was observed for all three enzymes if a particular element of structural symmetry was disrupted. For the BamHI MTase, integrity of the ATCC was critical, while an intact GAT sequence was necessary for the activity of T4Dam, and an intact GA was necessary for EcoDam. Theoretical alignment of the region of best contacts between the protein and DNA showed that in the case of a palindromic interaction site, a zone covering the 5′-symmetric residues is located in the major groove versus a zone of contact covering the 3′-symmetric residues in the minor groove. Our data fit a simple rule of thumb that the most important contacts are aligned around the methylation target base: if the target base is in the 5′ half of the palindrome, the interaction between the enzyme and the DNA occurs mainly in the major groove; if it is in the 3′ half, the interaction occurs mainly in the minor groove.
DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and anti-discriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.
Transcription of the bacteriophage Mu mom operon requires transactivation by the phage-encoded C protein. DNase I footprinting showed that in the absence of C, Escherichia coli RNA polymerase E(sigma)70 (RNAP) binds to the mom promoter (Pmom) region at a site, P2 (from -64 to -11 with respect to the transcription start site), on the top (non-transcribed) strand. This is slightly upstream from, but overlapping P1 (-49 to +16), the functional binding site for rightward transcription. Host DNA-[N6-adenine] methyltransferase (Dam) methylation of three GATCs immediately upstream of the C binding site is required to prevent binding of the E.coli OxyR protein, which represses mom transcription in dam- strains. OxyR, known to induce DNA bending, is normally in a reduced conformation in vivo, but is converted to an oxidized state under standard in vitro conditions. Using DNase I footprinting, we provide evidence supporting the proposal that the oxidized and reduced forms of OxyR interact differently with their target DNA sequences in vitro. A mutant form, OxyR-C199S, was shown to be able to repress mom expression in vivo in a dam- host. In vitro DNase I footprinting showed that OxyR-C199S protected Pmom from -104 to -46 on the top strand and produced a protection pattern characteristic of reduced wild-type OxyR. Prebinding of OxyR-C199S completely blocked RNAP binding to P2 (in the absence of C), whereas it only slightly decreased binding of C to its target site (-55 to -28, as defined by DNase I footprinting). In contrast, OxyR-C199S strongly inhibited C-activated recruitment of RNAP to P1. These results indicate that OxyR repression is mediated subsequent to binding by C. Mutations have been isolated that relieve the dependence on C activation and have the same transcription start site as the C-activated wild-type promoter. One such mutant, tin7, has a single base change at -14, which changes a T6 run to T3GT2. OxyR-C199S partially inhibited RNAP binding to the tin7 promoter in vitro, even though the OxyR and RNAP-P1 binding sites probably do not overlap, and in vivo expression of tin7 was reduced 5- to 10-fold in dam- cells. These results suggest that OxyR can repress tin7.
Type VI secretion systems (T6SS) are macromolecular machines of the cell envelope of Gram-negative bacteria responsible for bacterial killing and/or virulence towards different host cells. Here, we characterized the regulatory mechanism underlying expression of the enteroagregative Escherichia coli sci1 T6SS gene cluster. We identified Fur as the main regulator of the sci1 cluster. A detailed analysis of the promoter region showed the presence of three GATC motifs, which are target of the DNA adenine methylase Dam. Using a combination of reporter fusion, gel shift, and in vivo and in vitro Dam methylation assays, we dissected the regulatory role of Fur and Dam-dependent methylation. We showed that the sci1 gene cluster expression is under the control of an epigenetic switch depending on methylation: fur binding prevents methylation of a GATC motif, whereas methylation at this specific site decreases the affinity of Fur for its binding box. A model is proposed in which the sci1 promoter is regulated by iron availability, adenine methylation, and DNA replication.
DNA methylation plays an important role in the regulation of genes involved in assembly of cell surface adhesins or appendages. Methylation at a GATC motif by the Dam methylase influences binding of transcriptional regulators, leading to variation in the gene expression pattern. In several cases, this may lead to different cell subpopulations allowing a rapid adaptation to varying environments. In this work, we uncover the regulatory mechanism controlling expression of the sci1 Type VI secretion gene cluster in entero-aggregative Escherichia coli, which encodes a structure required for inter-bacterial interaction. We showed that this gene cluster is repressed by Fur in iron-replete conditions and that Fur binding on the promoter prevents methylation of a GATC motif. In iron-limited conditions, Fur is relieved from the promoter allowing expression of the gene cluster and methylation of the GATC motif. Methylation prevents de novo Fur binding allowing constitutive expression. Our findings support a model in which the expression of the Type VI secretion gene cluster is regulated by a non-stochastic epigenetic switch: switch from the OFF to ON phases depends on iron availability whereas the ON to OFF switch depends on DNA replication and competition between Dam-dependent methylation and Fur binding.
Most of the adenine residues in GATC sequences in the Escherichia coli chromosome are methylated by the enzyme deoxyadenosine methyltransferase (Dam). However, at least 20 GATC sequences remain nonmethylated throughout the cell cycle. Here we examined how the DNA methylation patterns of GATC sequences within the regulatory regions of the pyelonephritis-associated pilus (pap) operon and the glucitol utilization (gut) operon were formed. The results obtained with an in vitro methylation protection assay showed that the addition of the leucine-responsive regulatory protein (Lrp) to pap DNA was sufficient to protect the two GATC sequences in the pap regulatory region, GATC-I and GATC-II, from methylation by Dam. This finding was consistent with previously published data showing that Lrp was essential for methylation protection of these DNA sites in vivo. Methylation protection also occurred at a GATC site (GATC-44.5) centered 44.5 bp upstream of the transcription start site of the gutABD operon. Two proteins, GutR and the catabolite gene activator protein (CAP), bound to DNA sites overlapping the GATC-44.5-containing region of the gutABD operon. GutR, an operon-specific repressor, was essential for methylation protection in vivo, and binding of GutR protected GATC-44.5 from methylation in vitro. In contrast, binding of CAP at a site overlapping GATC-44.5 did not protect this site from methylation. Mutational analyses indicated that gutABD gene regulation was not controlled by methylation of GATC-44.5, in contrast to regulation of Pap pilus expression, which is directly controlled by methylation of the pap GATC-I and GATC-II sites.
The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.
DNA adenine methylation by DNA adenine methyltransferase (Dam) in Escherichia coli plays an important role in processes such as DNA replication initiation, gene expression regulation, and mismatch repair. In addition, E. coli strains deficient in Dam are hypersensitive to DNA-damaging agents. We used genome microarrays to compare the transcriptional profiles of E. coli strains deficient in Dam and mismatch repair (dam, dam mutS, and mutS mutants). Our results show that >200 genes are expressed at a higher level in the dam strain, while an additional mutation in mutS suppresses the induction of many of the same genes. We also show by microarray and semiquantitative real-time reverse transcription-PCR that both dam and dam mutS strains show derepression of LexA-regulated SOS genes as well as the up-regulation of other non-SOS genes involved in DNA repair. To correlate the level of SOS induction and the up-regulation of genes involved in recombinational repair with the level of DNA damage, we used neutral single-cell electrophoresis to determine the number of double-strand breaks per cell in each of the strains. We find that dam mutant E. coli strains have a significantly higher level of double-strand breaks than the other strains. We also observe a broad range in the number of double-strand breaks in dam mutant cells, with a minority of cells showing as many as 10 or more double-strand breaks. We propose that the up-regulation of recombinational repair in dam mutants allows for the efficient repair of double-strand breaks whose formation is dependent on functional mismatch repair.
The DNA of bacteriophage Mu, extracted from induced lysates, is partially resistant to digestion by the endonuclease BalI. This modification of DNA is controlled by the Mu modification function (mom), which acts in conjunction with the dam (DNA-adenine methylation) function of Escherichia coli. Since the BalI recognition site is apparently different from the dam recognition site, these results imply that either the specificity of the dam function is changed by the mom function or the mom function requires the dam function for its activity.
The dam gene of Escherichia coli encodes a DNA methyltransferase that methylates the N6 position of adenine in the sequence GATC. It was stably expressed from a shuttle vector in a repair- and recombination-proficient strain of Bacillus subtilis. In this strain the majority of plasmid DNA molecules was modified at dam sites whereas most chromosomal DNA remained unmethylated during exponential growth. During stationary phase the amount of unmethylated DNA increased, suggesting that methylated bases were being removed. An ultraviolet damage repair-deficient mutant (uvrB) contained highly methylated chromosomal and plasmid DNA. High levels of Dam methylation were detrimental to growth and viability of this mutant strain and some features of the SOS response were also induced. A mutant defective in the synthesis of adaptive DNA alkyltransferases and induction of the adaptive response (ada) also showed high methylation and properties similar to that of the dam gene expressing uvrB strain. When protein extracts from B. subtilis expressing the Dam methyltransferase or treated with N-methyl-N'-nitro-N-nitroso-guanidine were incubated with [3H]-labelled Dam methylated DNA, the methyl label was bound to two proteins of 14 and 9 kD. Some free N6-methyladenine was also detected in the supernatant of the incubation mixture. We propose that N6-methyladenine residues are excised by proteins involved in both excision (uvrB) and the adaptive response (ada) DNA repair pathways in B. subtilis.
Bacteria produce a variety of enzymes capable of methylating DNA. In many species, the majority of adenine methylation is accomplished by the DNA adenine methylase Dam. In Escherichia coli the Dam methylase plays roles in the initiation of replication, mismatch repair, and gene regulation. In a number of other bacterial species, mutation or overexpression of Dam leads to attenuation of virulence. Homologues of the dam gene exist in some members of the Firmicutes, including Streptococcus mutans, a dental pathogen. An S. mutans strain inactivated in the dam gene (SMU.504; here designated damA) was engineered, and phenotypes linked to cariogenicity were examined. A prominent observation was that the damA mutant produced greater amounts of glucan than the parental strain. Real-time PCR confirmed upregulation of gtfB. To determine whether other loci were affected by the damA mutation, a microarray analysis was carried out. Seventy genes were upregulated at least 2-fold in the damA mutant, and 33 genes were downregulated at least 2-fold. In addition to gtfB (upregulated 2.6-fold; 1.7-fold when measured by real-time PCR), other upregulated virulence factors included gbpC (upregulated 2.1-fold) and loci predicted to encode bacteriocins (upregulated 2- to 7-fold). Various sugar transport operons were also upregulated, the most extreme being the cellobiose operon (upregulated nearly 40-fold). Expression of sacB, encoding fructosyltransferase, was downregulated 2.4-fold. The sequence 5′-GATC-3′ appeared to constitute the recognition sequence for methylation. These results provide evidence that DNA methylation in S. mutans has a global effect on gene expression, including that of genes associated with cariogenic potential.
Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.
Properties of a mutant bacteriophage T2 DNA [N6-adenine] methyltransferase
(T2 Dam MTase) have been investigated for its potential utilization
in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state
kinetic analyses with oligonucleotide duplexes revealed that, compared
to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S
had a 1.5-fold higher kcat in methylating
canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies
in methylation of non-canonical GAY sites relative to the wild-type
enzymes. In agreement with these steady-state kinetic data, when
bacteriophage λ DNA was used as a substrate,
maximal protection from restriction nuclease cleavage in
vitro was achieved on the sequences GATC, GATN and GACY, while
protection of GACR sequences was less efficient. Collectively, our
data suggest that T2 Dam P126S can modify 28 recognition sequences.
The feasibility of using the mutant enzyme in RARE cleavage with BclI and EcoRV endonucleases has been
shown on phage λ DNA and with BclI
and DpnII endonucleases on yeast chromosomal DNA embedded
Phase variation of the outer membrane protein Ag43 in E. coli requires deoxyadenosine methylase (Dam) and OxyR. Previously, it was shown that OxyR is required for repression of the Ag43-encoding gene, agn43, and that Dam-dependent methylation of three GATC target sequences in the regulatory region abrogates OxyR binding. Here we report further characterization of agn43 transcription and its regulation. Transcription was initiated from a σ70-dependent promoter at the G residue of the upstream GATC sequence. Template DNA and RNA polymerase were sufficient to obtain transcription in vitro, but DNA methylation enhanced the level of transcription. Analyses of transcription in vivo of agn′-lacZ with mutated Dam target sequences support this conclusion. Since methylation also abrogates OxyR binding, this indicates that methylation plays a dual role in facilitating agn43 transcription. In vitro transcription from an unmethylated template was repressed by OxyR(C199S), which resembles the reduced form of OxyR. Consistent with this and the role of Dam in OxyR binding, OxyR(C199S) protected from DNase I digestion the agn43 regulatory region from −16 to +42, which includes the three GATC sequences. Deletion analyses of the regulatory region showed that a 101-nucleotide region of the agn43 regulatory region containing the promoter and this OxyR binding region was sufficient for Dam- and OxyR-dependent phase variation
Like many eukaryotes, bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Unlike eukaryotes, however, bacteria use DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation plays roles in the virulence of diverse pathogens of humans and livestock animals, including pathogenic Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine at GANTC sites by the CcrM methylase regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation at GATC sites by the Dam methylase provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage genomes, transposase activity, and regulation of gene expression. Transcriptional repression by Dam methylation appears to be more common than transcriptional activation. Certain promoters are active only during the hemimethylation interval that follows DNA replication; repression is restored when the newly synthesized DNA strand is methylated. In the E. coli genome, however, methylation of specific GATC sites can be blocked by cognate DNA binding proteins. Blockage of GATC methylation beyond cell division permits transmission of DNA methylation patterns to daughter cells and can give rise to distinct epigenetic states, each propagated by a positive feedback loop. Switching between alternative DNA methylation patterns can split clonal bacterial populations into epigenetic lineages in a manner reminiscent of eukaryotic cell differentiation. Inheritance of self-propagating DNA methylation patterns governs phase variation in the E. coli pap operon, the agn43 gene, and other loci encoding virulence-related cell surface functions.
DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics.
Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.
The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.
The effect of the number and position of DNA adenine methylation (dam) sites, i.e., d(GATC) sequences, on mismatch repair in Escherichia coli was investigated. The efficiency of repair was measured in an in vitro assay which used an f1 heteroduplex containing a G/T mismatch within the single EcoRI site. Both an increase in the number of dam sites and a shortened distance between dam site and mismatched site increased the efficiency of mismatch repair. The sequences adjacent to d(GATC) also affected the efficiency of methylation-directed mismatch repair. Furthermore, heteroduplexes with one extra dam site located close to either the 5' or 3' end of the excised base increased the repair efficiency to about the same extent. The findings suggest that the mismatch repair pathway has no preferred polarity.
The DNA-[N 6-adenine]-methyltransferase (Dam MTase) of phage T4 catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to the N6-position of adenine in the palindromic sequence, GATC. We have used a gel shift assay to monitor complex formation between T4 Dam and various synthetic duplex oligonucleotides, either native or modified/defective. The results are summarized as follows. (i) T4 Dam bound with approximately 100-fold higher affinity to a 20mer specific (GATC-containing) duplex containing the canonical palindromic methylation sequence, GATC, than to a non-specific duplex containing another palindrome, GTAC. (ii) Compared with the unmethylated duplex, the hemimethylated 20mer specific duplex had a slightly increased ( approximately 2-fold) ability to form complexes with T4 Dam. (iii) No stable complex was formed with a synthetic 12mer specific (GATC-containing) duplex, although T4 Dam can methylate it. This indicates that there is no relation between formation of a catalytically competent 12mer-Dam complex and one stable to gel electrophoresis. (iv) Formation of a stable complex did not require that both strands be contiguous or completely complementary. Absence of a single internucleotide phosphate strongly reduced complex formation only when missing between the T and C residues. This suggests that if T4 Dam makes critical contact(s) with a backbone phosphate(s), then the one between T and C is the only likely candidate. Having only one half of the recognition site intact on one strand was sufficient for stable complex formation provided that the 5'G.C base-pairs be present at both ends of the palindromic, GATC. Since absence of either a G or C abolished T4 Dam binding, we conclude that both strands are recognized by T4 Dam.
Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to Dam for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation sensitive digestion, cloning, and sequencing. Fifteen novel HCS binding sites were identified in the two cell lines and the following four out of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.
chromatin; DNA adenine methyl transferase; holocarboxylase synthetase; mammary cells
The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 Å resolution in the presence of S-adenosyl-l-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3′) half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E. coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-l-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.
Dam methylation; GATC recognition; base flipping; bacterial virulence factor