The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5’-amino-5’-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine (m5C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of m5C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung+ host proficient in uracil excision repair.
The ability to target DNA methylation toward a single, user-designated CpG site in vivo may have wide applicability for basic biological and biomedical research. A tool for targeting methylation toward single sites could be used to study the effects of individual methylation events on transcription, protein recruitment to DNA, and the dynamics of such epigenetic alterations. Although various tools for directing methylation to promoters exist, none offers the ability to localize methylation solely to a single CpG site. In our ongoing research to create such a tool, we have pursued a strategy employing artificially bifurcated DNA methyltransferases; each methyltransferase fragment is fused to zinc finger proteins with affinity for sequences flanking a targeted CpG site for methylation. We sought to improve the targeting of these enzymes by reducing the methyltransferase activity at non-targeted sites while maintaining high levels of activity at a targeted site. Here we demonstrate an in vitro directed evolution selection strategy to improve methyltransferase specificity and use it to optimize an engineered zinc finger methyltransferase derived from M.SssI. The unusual restriction enzyme McrBC is a key component of this strategy and is used to select against methyltransferases that methylate multiple sites on a plasmid. This strategy allowed us to quickly identify mutants with high levels of methylation at the target site (up to ∼80%) and nearly unobservable levels of methylation at a off-target sites (<1%), as assessed in E. coli. We also demonstrate that replacing the zinc finger domains with new zinc fingers redirects the methylation to a new target CpG site flanked by the corresponding zinc finger binding sequences.
Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methylation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins. The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5–52 nt from single zinc-factor binding sites. Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA. Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure. These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins. Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.
We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.
N.ϕGamma is a strand-specific and site-specific DNA nicking enzyme (YCG↓GT or AC↑CGR). Here we describe the isolation of single and double mutants of N.ϕGamma with attenuated activity. The nicking domains (NDs) of E59A and 11 double mutants were fused to the 5mCG-binding domain of MBD2 and generated fusion enzymes that preferentially nick 5mCG-modified DNA. The CG dinucleotide can be modified by C5 methyltransferases (MTases) such as M.SssI, M.HhaI or M.HpaII to create composite sites AC↑YGG N(8–15) 5mCG. We also constructed a fusion enzyme 2xMBD2-ND(N.BceSVIII) targeting more frequent composite sites AS↑YS N(5–12) 5mCG in Mn2+ buffer. 5mCG-dependent nicking requires special digestion conditions in high salt (0.3 M KCl) or in Ni2+ buffer. The fusion enzyme can be used to nick and label 5mCG-modified plasmid and genomic DNAs with fluorescently labeled Cy3-dUTP and potentially be useful for diagnostic applications, DNA sequencing and optical mapping of epigenetic markers. The importance of the predicted catalytic residues D89, H90, N106 and H115 in N.ϕGamma was confirmed by mutagenesis. We found that the wild-type enzyme N.ϕGamma prefers to nick 5mCG-modified DNA in Ni2+ buffer even though the nicking activity is sub-optimal compared to the activity in Mg2+ buffer.
From the characterization of enzyme activities and the analysis
of genomic sequences, the complement of DNA methyltransferases (MTases)
possessed by the cyanobacterium Anabaena PCC 7120
has been deduced. Anabaena has nine DNA MTases.
Four are associated with Type II restriction enzymes (AvaI, AvaII, AvaIII and the newly recognized
inactive AvaIV), and five are not. Of the latter,
four may be classified as solitary MTases, those whose function lies
outside of a restriction/modification system. The group
is defined here based on biochemical and genetic characteristics.
The four solitary MTases, DmtA/M.AvaVI,
and DmtD/M.AvaIX, methylate at GATC, GGCC,
CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the
N4 position, but its sequence is more similar to N6-adenine MTases
than to cytosine-specific enzymes, indicating that it may have evolved from
the former. The solitary MTases, appear to be of ancient origin
within cyanobacteria, while the restriction MTases appear to have
arrived by recent horizontal transfer as did five now inactive Type
I restriction systems. One Mtase, M.AvaV, cannot
reliably be classified as either a solitary or restriction MTase.
It is structurally unusual and along with a few proteins of prokaryotic
and eukaryotic origin defines a structural class of MTases distinct
from all previously described.
It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.
HP0593 DNA-(N6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5′-GCAG-3′and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.
Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.
Escherichia coli has two DNA repair methyltransferases (MTases): the 39-kilodalton (kDa) Ada protein, which can undergo proteolysis to an active 19-kDa fragment, and the 19-kDa DNA MTase II. We characterized DNA MTase II in cell extracts of an ada deletion mutant and compared it with the purified 19-kDa Ada fragment. Like Ada, DNA MTase II repaired O6-methylguanine (O6MeG) lesions via transfer of the methyl group from DNA to a cysteine residue in the MTase. Substrate competition experiments indicated that DNA MTase II repaired O4-methylthymine lesions by transfer of the methyl group to the same active site within the DNA MTase II molecule. The repair kinetics of DNA MTase II were similar to those of Ada; both repaired O6MeG in double-stranded DNA much more efficiently than O6MeG in single-stranded DNA. Chronic pretreatment of ada deletion mutants with sublethal (adapting) levels of two alkylating agents resulted in the depletion of DNA MTase II. Thus, unlike Ada, DNA MTase II did not appear to be induced in response to chronic DNA alkylation at least in this ada deletion strain. DNA MTase II was much more heat labile than Ada. Heat lability studies indicated that more than 95% of the MTase in unadapted E. coli was DNA MTase II. We discuss the possible implications of these results for the mechanism of induction of the adaptive response. A similarly active 19-kDa O6MeG-O4-methylthymine DNA MTase was identified in Salmonella typhimurium.
Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.
The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.
Genetic transformation of bacteria harboring multiple Restriction-Modification (R-M) systems is often difficult using conventional methods. Here, we describe a mimicking-of-DNA-methylation-patterns (MoDMP) pipeline to address this problem in three difficult-to-transform bacterial strains. Twenty-four putative DNA methyltransferases (MTases) from these difficult-to-transform strains were cloned and expressed in an Escherichia coli strain lacking all of the known R-M systems and orphan MTases. Thirteen of these MTases exhibited DNA modification activity in Southwestern dot blot or Liquid Chromatography–Mass Spectrometry (LC–MS) assays. The active MTase genes were assembled into three operons using the Saccharomyces cerevisiae DNA assembler and were co-expressed in the E. coli strain lacking known R-M systems and orphan MTases. Thereafter, results from the dot blot and restriction enzyme digestion assays indicated that the DNA methylation patterns of the difficult-to-transform strains are mimicked in these E. coli hosts. The transformation of the Gram-positive Bacillus amyloliquefaciens TA208 and B. cereus ATCC 10987 strains with the shuttle plasmids prepared from MoDMP hosts showed increased efficiencies (up to four orders of magnitude) compared to those using the plasmids prepared from the E. coli strain lacking known R-M systems and orphan MTases or its parental strain. Additionally, the gene coding for uracil phosphoribosyltransferase (upp) was directly inactivated using non-replicative plasmids prepared from the MoDMP host in B. amyloliquefaciens TA208. Moreover, the Gram-negative chemoautotrophic Nitrobacter hamburgensis strain X14 was transformed and expressed Green Fluorescent Protein (GFP). Finally, the sequence specificities of active MTases were identified by restriction enzyme digestion, making the MoDMP system potentially useful for other strains. The effectiveness of the MoDMP pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate the functional genomics of the strains that are difficult to transform.
Approximately 95% of the genome-sequenced bacteria harbor Restriction-Modification (R-M) systems. R-M systems usually occur in pairs, i.e., DNA methyltransferases (MTases) and restriction endonucleases (REases). REases can degrade invading DNA to protect the cell from infection by phages. This protecting machinery has also become the barrier for experimental genetic manipulation, because the newly introduced DNA would be degraded by the REases of the transformed bacteria. In this study we have developed a pipeline to protect DNA by methylation from cleavage by host REases. Multiple DNA MTases were cloned from three difficult-to-transform bacterial strains and co-expressed in an E. coli strain lacking all of the known endogenous R-M systems and orphan MTases. Thus, the DNA methylation patterns of these strains have become similar to that of the difficult-to-transform strains. Ultimately, the DNA prepared from these E. coli strains can overcome the R-M barrier of the bacterial strains that are difficult to transform and achieve genetic manipulation. The effectiveness of this pipeline in different bacterial groups suggests a universal potential. This pipeline could facilitate functional genomics of bacterial strains that are difficult to transform.
DNA methyltransferases (MTases), unlike MTases acting on other substrates, exhibit sequence permutation. Based on the sequential order of the cofactor-binding subdomain, the catalytic subdomain, and the target recognition domain (TRD), several classes of permutants have been proposed. The majority of known DNA MTases fall into the α, β, and γ classes. There is only one member of the ζ class known and no members of the δ and ε classes have been identified to date. Two mechanisms of permutation have been proposed: one involving gene duplication and in-frame fusion, and the other involving inter- and intragenic shuffling of gene segments.
Two novel cases of sequence permutation in DNA MTases implicated in restriction-modification systems have been identified, which suggest that members of the δ and ζ classes (M.MwoI and M.TvoORF1413P, respectively) evolved from β-class MTases. This is the first identification of the δ-class MTase and the second known ζ-class MTase (the first ζ-class member among DNA:m4C and m6A-MTases).
Fragmentation of a DNA MTase gene may result from attack of nucleases, for instance when the RM system invades a new cell. Its reassembly into a functional form, the order of motifs notwithstanding, may be strongly selected for, if the cognate ENase gene remains active and poses a threat to the host's chromosome. The "cut-and-paste" mechanism is proposed for β-δ permutation, which is non-circular and involves relocation of one segment of a gene. The circular β-ζ permutation may be explained both by gene duplication or shuffling of gene fragments. These two mechanisms are not mutually exclusive and probably both played a role in the evolution of permuted DNA MTases.
We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase). This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa. The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells. Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain. The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases. The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases. A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung. DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library. The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization. Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.
Little is known about the effects of single DNA methylation events on gene transcription. The ability to direct the methylation toward a single unique site within a genome would have broad use as a tool to study the effects of specific epigenetic changes on transcription. A targeted enzyme might also be useful in a therapy for diseases with an epigenetic component or as a means to site-specifically label DNA. Previous studies have sought to target methyltransferase activity by fusing DNA binding proteins to methyltransferases. However, the methyltransferase domain remains active even when the DNA binding protein is unbound, resulting in significant off-target methylation. A better strategy would make methyltransferase activity contingent upon the DNA binding protein’s association with its DNA binding site. We have designed targeted methyltransferases by fusing zinc fingers to the fragments of artificially-bisected, assembly-compromised methyltransferases. The zinc fingers’ binding sites flank the desired target site for methylation. Zinc finger binding localizes the two fragments near each other encouraging their assembly only over the desired site. Through a combination of molecular modeling and experimental optimization in E. coli, we created an engineered methyltransferase derived from M.HhaI with 50–60% methylation at a target site and nearly undetectable levels of methylation at a non-target M.HhaI site (1.4±2.4%). Using a restriction digestion assay, we demonstrate that localization of both fragments synergistically increases methylation at the target site, illustrating the promise of our approach.
This study creates a device where the DNA is electronically integrated to serve as both the biological target and electrical transducer in a CNT–DNA–CNT device. We detect DNA binding and methylation by the methyltransferase M.SssI at the single molecule level. We demonstrate sequence-specific, reversible binding of M.SssI and protein-catalyzed methylation that alters the protein-binding affinity of the device. This device, which relies on the exquisite electrical sensitivity of DNA, represents a unique route for the specific, single molecule detection of enzymatic activity.
A real-time assay for CpG-specific cytosine-C5 methyltransferase activity has been developed. The assay applies a break light oligonucleotide in which the methylation of an unmethylated 5′-CG-3′ site is enzymatically coupled to the development of a fluorescent signal. This sensitive assay can measure rates of DNA methylation down to 0.34 ± 0.06 fmol/s. The assay is reproducible, with a coefficient of variation over six independent measurements of 4.5%. Product concentration was accurately measured from fluorescence signals using a linear calibration curve, which achieved a goodness of fit (R2) above 0.98. The oligonucleotide substrate contains three C5-methylated cytosine residues and one unmethylated 5′-CG-3′ site. Methylation yields an oligonucleotide containing the optimal substrate for the restriction enzyme GlaI. Cleavage of the fully methylated oligonucleotide leads to separation of fluorophore from quencher, giving a proportional increase in fluorescence. This method has been used to assay activity of DNMT1, the principle maintenance methyltransferase in human cells, and for the kinetic characterization of the bacterial cytosine-C5 methyltransferase M.SssI. The assay has been shown to be suitable for the real-time monitoring of DNMT1 activity in a high-throughput format, with low background signal and the ability to obtain linear rates of methylation over long periods, making this a promising method of high-throughput screening for inhibitors.
The ability to target methylation to specific genomic sites would further the study of DNA methylation’s biological role and potentially offer a tool for silencing gene expression and for treating diseases involving abnormal hypomethylation. The end-to-end fusion of DNA methyltransferases to zinc fingers has been shown to bias methylation to desired regions. However, the strategy is inherently limited because the methyltransferase domain remains active regardless of whether the zinc finger domain is bound at its cognate site and can methylate non-target sites. We demonstrate an alternative strategy in which fragments of a DNA methyltransferase, compromised in their ability to methylate DNA, are fused to two zinc fingers designed to bind 9 bp sites flanking a methylation target site. Using the naturally heterodimeric DNA methyltransferase M.EcoHK31I, which methylates the inner cytosine of 5′-YGGCCR-3′, we demonstrate that this strategy can yield a methyltransferase capable of significant levels of methylation at the target site with undetectable levels of methylation at non-target sites in Escherichia coli. However, some non-target methylation could be detected at higher expression levels of the zinc finger methyltransferase indicating that further improvements will be necessary to attain the desired exclusive target specificity.
A type IIG restriction endonuclease, RM.BpuSI from Bacillus pumilus, has been characterized and its X-ray crystal structure determined at 2.35Å resolution. The enzyme is comprised of an array of 5-folded domains that couple the enzyme's N-terminal endonuclease domain to its C-terminal target recognition and methylation activities. The REase domain contains a PD-x15-ExK motif, is closely superimposable against the FokI endonuclease domain, and coordinates a single metal ion. A helical bundle domain connects the endonuclease and methyltransferase (MTase) domains. The MTase domain is similar to the N6-adenine MTase M.TaqI, while the target recognition domain (TRD or specificity domain) resembles a truncated S subunit of Type I R–M system. A final structural domain, that may form additional DNA contacts, interrupts the TRD. DNA binding and cleavage must involve large movements of the endonuclease and TRD domains, that are probably tightly coordinated and coupled to target site methylation status.
Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.
RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced. This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI. The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da. A fragment of R. sphaeroides chromosomal DNA exhibited M.RsrI activity in E. coli and was used to sequence the rsrIM gene. The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15. In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology. Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes. The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl. Acids Res. (1989) 17, this issue).
Many flaviviruses are globally important human pathogens. Their plus-strand RNA genome contains a 5′-cap structure that is methylated at the guanine N-7 and the ribose 2′-OH positions of the first transcribed nucleotide, adenine (m7GpppAm). Using West Nile virus (WNV), we demonstrate, for the first time, that the nonstructural protein 5 (NS5) mediates both guanine N-7 and ribose 2′-O methylations and therefore is essential for flavivirus 5′-cap formation. We show that a recombinant full-length and a truncated NS5 protein containing the methyltransferase (MTase) domain methylates GpppA-capped and m7GpppA-capped RNAs to m7GpppAm-RNA, using S-adenosylmethionine as a methyl donor. Furthermore, methylation of GpppA-capped RNA sequentially yielded m7GpppA- and m7GpppAm-RNA products, indicating that guanine N-7 precedes ribose 2′-O methylation. Mutagenesis of a K61-D146-K182-E218 tetrad conserved in other cellular and viral MTases suggests that NS5 requires distinct amino acids for its N-7 and 2′-O MTase activities. The entire K61-D146-K182-E218 motif is essential for 2′-O MTase activity, whereas N-7 MTase activity requires only D146. The other three amino acids facilitate, but are not essential for, guanine N-7 methylation. Amino acid substitutions within the K61-D146-K182-E218 motif in a WNV luciferase-reporting replicon significantly reduced or abolished viral replication in cells. Additionally, the mutant MTase-mediated replication defect could not be trans complemented by a wild-type replicase complex. These findings demonstrate a critical role for the flavivirus MTase in viral reproduction and underscore this domain as a potential target for antiviral therapy.
Twenty AdoMet-dependent methyltransferases (MTases) have been
characterized structurally by X-ray crystallography and NMR. These
include seven DNA MTases, five RNA MTases, four protein MTases and four
small molecule MTases acting on the carbon, oxygen or nitrogen atoms
of their substrates. The MTases share a common core structure of
a mixed seven-stranded β-sheet (6↓ 7↑ 5↓ 4↓ 1↓ 2↓ 3↓) referred to as an ‘AdoMet-dependent
MTase fold’, with the exception of a protein arginine MTase
which contains a compact consensus fold lacking the antiparallel
hairpin strands (6↓ 7↑).
The consensus fold is useful to identify hypothetical MTases during structural
proteomics efforts on unannotated proteins. The same core structure
works for very different classes of MTase including those that act
on substrates differing in size from small molecules (catechol or
glycine) to macromolecules (DNA, RNA and protein). DNA MTases use
a ‘base flipping’ mechanism to deliver a specific
base within a DNA molecule into a typically concave catalytic pocket. Base
flipping involves rotation of backbone bonds in double-stranded
DNA to expose an out-of-stack nucleotide, which can then be a substrate
for an enzyme-catalyzed chemical reaction. The phenomenon is fully
established for DNA MTases and for DNA base excision repair enzymes,
and is likely to prove general for enzymes that require access to
unpaired, mismatched or damaged nucleotides within base-paired regions
in DNA and RNA. Several newly discovered MTase families in eukaryotes
(DNA 5mC MTases and protein arginine and lysine MTases) offer new challenges
in the MTase field.
Many flaviviruses are significant human pathogens. No effective antiviral therapy is currently available for treatment of flavivirus infections. Development of antiviral treatment against these viruses is urgently needed. The flavivirus methyltransferase (MTase) responsible for N-7 and 2'-O methylation of the viral RNA cap has recently been mapped to the N-terminal region of nonstructural protein 5. Structural and functional studies suggest that the MTase represents a novel antiviral target. Here we review current understanding of flavivirus RNA cap methylation and its implications for development of antivirals. The 5' end of the flavivirus plus-strand RNA genome contains a type 1 cap structure (m7GpppAmG). Flaviviruses encode a single MTase domain that catalyzes two sequential methylations of the viral RNA cap, GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA, using S-adenosyl-L-methionine (SAM) as the methyl donor. The two reactions require different viral RNA elements and distinct biochemical assay conditions. Despite exhibiting two distinct methylation activities, flavivirus MTase contains a single binding site for SAM in its crystal structure. Therefore, substrate GpppA-RNA must be re-positioned to accept the N-7 and 2'-O methyl groups from SAM during the two methylation reactions. Structure-guided mutagenesis studies indeed revealed two distinct sets of amino acids on the enzyme surface that are specifically required for N-7 and 2'-O methylation. In the context of virus, West Nile viruses (WNV) defective in N-7 methylation are non-replicative; however, WNVs defective in 2'-O methylation are attenuated and can protect mice from subsequent wild-type WNV challenge. Collectively, the results demonstrate that the N-7 MTase represents a novel target for flavivirus therapy.
Flavivirus NS5; Methyltransferase; Flavivirus replication; Antiviral therapy; West Nile virus; dengue virus; yellow fever virus; Japanese encephalitis virus; tick-borne encephalitis virus