GATC Sequences Research Articles

Cloning, sequence analysis and heterologous expression of the DNA adenine-(N(6)) methyltransferase from the human pathogen Actinobacillus actinomycetemcomitans.

We cloned and sequenced the DNA adenine-N(6) methyltransferase gene of the human pathogen Actinobacillus actinomycetemcomitans (M.AacDAM). Restriction digestion shows that the enzyme methylates adenine in the sequence GATC. Expression of the enzyme in a DAM(-) background shows in vivo activity. A PSI-BLAST search revealed that M.AacDAM is most related to M.HindIV, M.EcoDAM, M.StyDAM, and M.SmaII. The ClustalW alignment shows highly conserved regions in the enzyme characteristic for type a MTases. Phylogenetic tree analysis shows a cluster of enzymes recognizing the sequence GATC, within a branch of orphan MTases harboring M.AacDAM. The cloning and sequencing of this first methyltransferase gene described for A. actinomycetemcomitans open the path for studies on the potential regulatory impact of DNA methylation on gene regulation and virulence in this organism.

A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation.

SeqA is an Escherichia coli DNA-binding protein that acts at replication origins and controls DNA replication. However, binding is not exclusive to origins. Many fragments containing two or more hemi-methylated GATC sequences bind efficiently. Binding was optimal when two such sequences were closely apposed or up to 31 bases apart on the same face of the DNA helix. Binding studies suggest that neighboring bound proteins contact each other to form a complex with the intervening DNA looped out. There are many potential binding sites distributed around the E.coli chromosome. As replication produces a transient wave of hemi-methylation, tracts of SeqA binding are likely to associate with each fork as replication progresses. The number and positions of green fluorescent protein-SeqA foci seen in living cells suggest that they correspond to these tracts, and that the forks are tethered to planes of cell division. SeqA may help to tether the forks or to organize newly replicated DNA into a structure that aids DNA to segregate away from the replication machinery.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Pre-steady state kinetics of bacteriophage T4 dam DNA-[N(6)-adenine] methyltransferase: interaction with native (GATC) or modified sites.

The DNA methyltransferase of bacteriophage T4 (T4 Dam MTase) recognizes the palindromic sequence GATC, and catalyzes transfer of the methyl group from S:-adenosyl-L-methionine (AdoMet) to the N(6)-position of adenine [generating N(6)-methyladenine and S:-adenosyl-L-homocysteine (AdoHcy)]. Pre-steady state kinetic analysis revealed that the methylation rate constant k(meth) for unmethylated and hemimethylated substrates (0.56 and 0.47 s(-1), respectively) was at least 20-fold larger than the overall reaction rate constant k(cat) (0.023 s(-1)). This indicates that the release of products is the rate-limiting step in the reaction. Destabilization of the target-base pair did not alter the methylation rate, indicating that the rate of target nucleoside flipping does not limit k(meth). Preformed T4 Dam MTase-DNA complexes are less efficient than preformed T4 Dam MTase-AdoMet complexes in the first round of catalysis. Thus, this data is consistent with a preferred route of reaction for T4 Dam MTase in which AdoMet is bound first; this preferred reaction route is not observed with the DNA-[C5-cytosine]-MTases.

Bacterial DNA Methylation and Gene Transfer Efficiency

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial GmATC and CmC(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam−, dcm−) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam+, dcm+) or JM110 (dam−, dcm−) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam+, dcm+) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam−, dcm−) counterpart. Therefore, these data do not suggest that the use of (dam−, dcm−) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam+, dcm+) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial GmATC and CmC(A/T)GG sequences in eukaryotic cells or organisms.

A SeqA hyperstructure and its interactions direct the replication and sequestration of DNA.

A level of explanation in biology intermediate between macromolecules and cells has recently been proposed. This level is that of hyperstructures. One class of hyperstructures comprises the genes, mRNA, proteins and lipids that assemble to fulfil a particular function and disassemble when no longer required. To reason in terms of hyperstructures, it is essential to understand the factors responsible for their formation. These include the local concentration of sites on DNA and their cognate DNA-binding proteins. In Escherichia coli, the formation of a SeqA hyperstructure via the phenomenon of local concentration may explain how the binding of SeqA to hemimethylated GATC sequences leads to the sequestration of newly replicated origins of replication.

SNDV, a Novel Virus of the Extremely Thermophilic and Acidophilic Archaeon Sulfolobus

We describe a novel virus, SNDV (Sulfolobus neozealandicus droplet-shaped virus), of the crenarchaeotal archaeon Sulfolobus, which was found in a carrier state in a Sulfolobus strain isolated from a field sample from New Zealand. SNDV particles are droplet-shaped and densely covered by thin tail fibers at their pointed ends. The virion consists of a core and a coat. The latter has the appearance of a beehive and has a surface that is either helically ribbed or a stack of hoops. The genome is cccDNA of 20 kb, which is modified by dam-like methylation. It is cleaved by only a few type II restriction enzymes e.g., DpnI but not MboI, demonstrating an N(6)-methylation of the adenine residue in GATC sequences. The DNA-modifying system differentiates between virus and host. We postulate a virus-encoded methylase that is active on hemimethylated DNA. The host range of SNDV is confined to few Sulfolobus strains from New Zealand. The virus persists in an unstable carrier state rather than as a prophage. Due to its uniqueness we propose to assign it to a novel virus family termed Guttaviridae.

Lack of GATC sites in the genome of Streptococcus bovis bacteriophage F4

A strong bias against GATC sites was observed in the genome of phage F4, a lytic Streptococcus bovis bacteriophage. Only three GATC sites were found within the 60.4-kbp genome of this phage. The comparative lack of GATC sequences within the F4 genome was probably not due to dam methylation, as no modification within this site was detected using methylation-sensitive isoschizomer pair restriction endonuclease analysis. The short oligonucleotide composition of available S. bovis DNA sequences suggested the existence of an unknown mechanism for counterselection of GATC sites in S. bovis bacteriophages.

Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyR-mediated repression of transcription.

It has been shown previously that phase variation of the outer membrane protein Antigen43 (Ag43) of Escherichia coli requires the DNA-methylating enzyme deoxyadenosine methyltransferase (Dam) and the global regulator OxyR. In this study, we analysed the regulation of the Ag43 encoding gene (agn) using isolates containing a fusion of the agn regulatory region to the reporter gene lacZ. Our results indicate that phase variation of Ag43 is regulated at the level of transcription. Repression of agn'-lacZ transcription required OxyR, whereas activation required Dam. The regulatory region of agn contains three GATC sequences, which are target sites for Dam-dependent methylation. In vivo, the methylation state of these GATC sequences correlated with the transcription state of agn'-lacZ. These GATC sequences were not protected from Dam-dependent methylation in an oxyR background, suggesting that OxyR binding results in Dam-dependent methylation protection in OFF cells. In vitro, both oxidized OxyR and OxyR(C199S), which is locked in the reduced conformation, bound to the agn regulatory region, but methylation of the three GATC sequences abrogated this binding. In vivo, OxyR(C199S) was sufficient to repress Ag43 transcription. Our data support a model in which OxyR-mediated repression of transcription is alleviated by methylation of three GATC sequences in its binding site. In addition, we show that, in an oxyR background, Dam was still required for full activation, suggesting that the model concerning the role of Dam in agn regulation is incomplete. These results show that Dam-dependent phase variation in E. coli is not limited to the previously identified regulatory system of the family of pap-like fimbrial operons.

Asymmetry of Dam remethylation on the leading and lagging arms of plasmid replicative intermediates.

In Escherichia coli, adenine methylation at the sequence GATC allows coupling of cellular processes to chromosome replication and the cell cycle. The transient presence of hemimethylated DNA after replication facilitates post-replicative mismatch repair, induces transcription of some genes and allows transposition of mobile elements. We were interested in estimating the half-life of hemimethylated DNA behind the replication fork in plasmid molecules and in determining whether Dam methyltransferase restores N6 adenine methylation simultaneously on both replicative arms. We show that remethylation takes place asynchronously on the leading and lagging daughter strands shortly after replication. On the leading arm the fully methylated adenine is restored approximately 2000 bp (corresponding to 2 s) behind the replication fork, while remethylation takes twice as long (at 3500-4000 bp or approximately 3.5-4 s) on the lagging replicative arm. This observation suggests that Dam remethylation of the lagging arm requires ligated Okazaki fragments.

Synthetic DNA minor groove-binding drugs

In this review, both cationic and neutral synthetic ligands that bind in the minor groove of DNA are discussed. Certain bis-distamycins and related lexitropsins show activities against human immunodeficiency virus (HIV)-1 and HIV-2 at low nanomolar concentrations. DAPI binds strongly to AT-containing polymers and is located in the minor groove of DNA. DAPI intercalates in DNA sequences that do not contain at least three consecutive AT bp. Berenil can also exhibit intercalative, as well as minor groove binding, properties depending on sequence. Furan-containing analogues of berenil play an important role in their activities against Pneumocystis carinii and Cryptosporidium parvuam infections in vivo. Pt(II)-berenil conjugates show a good activity profile against HL60 and U-937 human leukemic cells. Pt-pentamidine shows higher antiproliferative activity against small cell lung, non-small cell lung, and melanoma cancer cell lines compared with many other tumor cell lines. trans-Butenamidine shows good anti- P. carinii activity in rats. Pentamidine is used against P. carinii pneumonia in individuals infected with HIV who are at high risk from this infection. A comparison of the cytotoxic potencies of adozelesin, bizelesin, carzelesin, cisplatin, and doxorubicin indicates that adozelesin is a potent analog of CC-1065. Naturally occurring pyrrolo[2,1- c][1,4]benzodiazepines such as anthramycin have a 2- to 3-bp sequence specificity, but a synthetic PBD dimer spans 6 bp, actively recognizing a central 5′-GATC sequence. The crosslinking efficiency of PBD dimers is much greater than that of other major groove crosslinkers, such as cisplatin, melphalan, etc. Neothramycin is used clinically for the treatment of superficial carcinoma of the bladder.

Binding of SeqA protein to DNA requires interaction between two or more complexes bound to separate hemimethylated GATC sequences.

The SeqA protein binds to the post-replicative forms of the origins of replication of the Escherichia coli chromosome (oriC) and the P1 plasmid (P1oriR) at hemimethylated GATC adenine methylation sites. It appears to regulate replication by preventing premature reinitiation. However, SeqA binding is not exclusive to replication origins: different fragments with hemimethylated GATC sites can bind SeqA in vitro when certain rules apply. Most notably, more than one such site must be present on a bound fragment. The protein appears to recognize individual hemimethylated sites, but must undergo an obligate cooperative interaction with a nearby bound protein for stable binding. SeqA contacts both DNA strands in a discrete patch at each hemimethylated GATC sequence. All four GATC bases are contacted and are essential for binding. Although the recognized sequence is symmetrical, the footprint on the methylated strand is always broader, suggesting that the bound protein is positioned asymmetrically with its orientation dictated by the position of the unique methyl group. Studies of alternative spacings and relative orientations of adjacent sites suggest that each site may be recognized by a symmetrical dimer with an induced asymmetry in one of the subunits similar to that seen with certain type II restriction endonucleases.

Interaction of SeqA and Dam Methylase on the Hemimethylated Origin of Escherichia coli Chromosomal DNA Replication

Preferential binding of SeqA protein to hemimethylated oriC, the origin of Escherichia coli chromosomal replication, delays methylation by Dam methylase. Because the SeqA-oriC interaction appears to be essential in timing of chromosomal replication initiation, the biochemical functions of SeqA protein and Dam methylase at the 13-mer L, M, and R region containing 4 GATC sequences at the left end of oriC were examined. We found that SeqA protein preferentially bound hemimethylated 13-mers but not fully nor unmethylated 13-mers. Regardless of strand methylation, the binding of SeqA protein to the hemimethylated GATC sequence of 13-mer L was followed by additional binding to other hemimethylated GATC sequences of 13-mer M and R. On the other hand, Dam methylase did not discriminate binding of 13-mers in different methylation patterns and was not specific to GATC sequences. The binding specificity and higher affinity of SeqA protein over Dam methylase to the hemimethylated 13-mers along with the reported cellular abundance of this protein explains the dominant action of SeqA protein over Dam methylase to the newly replicated oriC for the sequestration of chromosomal replication. Furthermore, SeqA protein bound to hemimethylated 13-mers was not dissociated by Dam methylase, and most SeqA protein spontaneously dissociated 10 min after binding. Also, SeqA protein delayed the in vitro methylation of hemimethylated 13-mers by Dam methylase. These in vitro results suggest that the intrinsic binding instability of SeqA protein results in release of sequestrated hemimethylated oriC.

The assembly and migration of SeqA-Gfp fusion in living cells of Escherichia coli.

SeqA protein, which binds to hemi-methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild-type Escherichia coli cells. In this work, we observed cellular localization of the SeqA-Gfp fusion in living cells. SeqA-Gfp was localized to a discrete focus or foci in wild-type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase. These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy. Time-lapse experiments revealed that duplicated SeqA-Gfp foci migrated rapidly in opposite directions. Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA-Gfp is biologically active. Immunoprecipitation of the fusion from cell extracts using anti-Gfp antibody indicated that the fusion was assembled with the wild-type SeqA protein.

Crystal structure of the DpnM DNA adenine methyltransferase from the DpnII restriction system of streptococcus pneumoniae bound to S-adenosylmethionine.

. Methyltransferases (Mtases) catalyze the transfer of methyl groups from S-adenosylmethionine (AdoMet) to a variety of small molecular and macromolecular substrates. These enzymes contain a characteristic alpha/beta structural fold. Four groups of DNA Mtases have been defined and representative structures have been determined for three groups. DpnM is a DNA Mtase that acts on adenine N6 in the sequence GATC; the enzyme represents group alpha DNA Mtases, for which no structures are known. . The structure of DpnM in complex with AdoMet was determined at 1.80 A resolution. The protein comprises a consensus Mtase fold with a helical cluster insert. DpnM binds AdoMet in a similar manner to most other Mtases and the enzyme contains a hollow that can accommodate DNA. The helical cluster supports a shelf within the hollow that may recognize the target sequence. Modeling studies indicate a potential site for binding the target adenine, everted from the DNA helix. Comparison of the DpnM structure and sequences of group alpha DNA Mtases indicates that the group is a genetically related family. Structural comparisons show DpnM to be most similar to a small-molecule Mtase and then to macromolecular Mtases, although several dehydrogenases show greater similarity than one DNA Mtase. . DpnM, and by extension the DpnM family or group alpha Mtases, contains the consensus fold and AdoMet-binding motifs found in most Mtases. Structural considerations suggest that macromolecular Mtases evolved from small-molecule Mtases, with different groups of DNA Mtases evolving independently. Mtases may have evolved from dehydrogenases. Comparison of these enzymes indicates that in protein evolution, the structural fold is most highly conserved, then function and lastly sequence.

Increased expression of a hemimethylated oriC binding protein, SeqA, in an aphA mutant

In Escherichia coli, the origin of DNA replication, oriC, becomes transiently hemimethylated at the GATC sequences immediately after initiation of replication and this hemimethylated state is prolonged because of its sequestration by a fraction of outer membrane. This sequestration is dependent on a hemimethylated oriC binding protein such as SeqA. We previously isolated a clone of phage λgtll called hobH, producing a LacZ fusion protein which recognizes hemimethylated oriC DNA. Very recently, Thaller et al. (FEMS Microbiol. Lett. 146 (1997)191–198)found that the same DNA segment encodes a non-specific acid phosphatase, and named the gene aphA. We show here that the interruption of the aphA reading frame by kanamycin resistance gene insertion, abolishes acid phosphatase (NAP) activity. Interestingly, in the membrane of the null mutant, the amount of SeqA protein is about six times higher than that in the parental strain, suggesting the existence of a regulatory mechanism between SeqA and NAP expression.

Formation of DNA methylation patterns: nonmethylated GATC sequences in gut and pap operons.

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.

Regulation of UropathogenicEscherichia coliAdhesin Expression by DNA Methylation

Pap pili play an important role in the pathogenesis of upper urinary tract infections by enabling uropathogenicEscherichia colito adhere to host epithelial cells. Pap pili are coded for by the pyelonephritis-associated pili (pap) operon, which consists of 11 genes required for the expression and assembly of Pap pili. Expression of Pap pili is regulated by a phase variation mechanism in which the pili expression state of the bacterial population is skewed between phase-on (expression positive) and phase-off (expression negative) states. Pap phase variation is controlled by the cooperative binding of leucine-responsive regulatory protein (Lrp) to two sets of Lrp binding sites in the upstream regulatory region of thepapoperon. A single GATC sequence, which is the target site for deoxyadenosine methylase (Dam), is centrally located within each Lrp binding region. Dam plays a critical role in the expression of Pap pili via the formation of DNA methylation patterns. Methylation of GATC-I reduced the affinity of Lrp forpapDNA by about twofold. Conversely, Lrp specifically blocked methylation ofpapGATC-Iin vitro.These data support the hypothesis that Lrp and Dam compete for binding to GATC-I, and are consistent with previous results indicating that methylation of GATC-I is important for stability of the phase-off state.

Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions.

A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein.

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at all oriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus, datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations when oriC is desequestered.