Biochemical and structural characterization of the first-discovered metazoan DNA cytosine-N4 methyltransferase from the bdelloid rotifer Adineta vaga.

Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication.

A novel signal-off photoelectrochemical biosensor for M.SssI MTase activity assay based on GQDs@ZIF-8 polyhedra as signal quencher.

DNA methylation catalyzed by M.SssI methyltransferases (MTase) has important roles in gene expression and other cellular activities, and relates to some diseases, especially cancers. Therefore, it is important to develop a sensitive sensing platform for M.SssI MTase activity assay. Here, taking zeolitic imidazolate framework-8 (ZIF-8) polyhedra as the carriers of graphene quantum dots (GQDs), GQDs-embedded ZIF-8 polyhedra (denoted as GQDs@ZIF-8 polyhedra) were successfully prepared and used as the multi-functional signal quencher to construct a novel signal-off photoelectrochemical (PEC) biosensor for M.SssI MTase activity assay. Firstly, the indium tin oxide (ITO) slice was modified with TiO2, poly(diallyldimethylammonium chloride) and CdTe quantum dots (QDs). The obtained electrode was used as the photoelectrode and labeled as ITO/TiO2/CdTe QDs. Then, single-stranded DNA (S1) was anchored on the photoelectrode surface via S-Cd bond. After hybridization between S1 and biotinylated single-stranded DNA (S2), the streptavidin (SA)-labeled GQDs@ZIF-8 polyhedra were introduced to the modified electrode via the specific reaction between biotin and SA. As the signal quencher, GQDs@ZIF-8 polyhedra could not only inhibit the photocurrent signal of the ITO/TiO2/CdTe QDs electrode due to the steric hindrance effect, but also act as peroxidase mimetics to catalyze precipitation reaction of 4-chloro-1-naphthol, resulting in the evident depression of the photocurrent signal. For the specially designed S1/S2 double-strand DNA, the decreased photocurrent was quantitatively correlated with the M.SssI MTase activity (linear response range, 0.005-150 U mL-1; detection limit, 0.004 U mL-1). The developed GQDs@ZIF-8 polyhedra and related PEC biosensor may have potential applications in clinical research and disease diagnosis.

Isolation and characterization of site-specific DNA-methyltransferases from Bacillus coagulans K.

Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'.

Recombinant alpha and beta subunits of M.AquI constitute an active DNA methyltransferase.

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit

Structure, function, and mechanism of HhaI DNA methyltransferases.

A vast amount of literature has accumulated on the characterization of DNA methyltransferases. The HhaI DNA methyltransferase, a C5-cytosine methyltransferase, has been the subject of investigation for the last 2 decades. Biochemical and kinetic characterization have led to an understanding of the catalytic and kinetic mechanism of the methyltransfer reaction. The HhaI methyltransferase has also been subjected to extensive structural analysis, with the availability of 12 structures with or without a cofactor and a variety of DNA substrates. The mechanism of base flipping, first described for the HhaI methyltransferase, is conserved among all DNA methyltransferases and is also found to occur in numerous DNA repair enzymes. Studies with other methyltransferase reveal a significant structural and functional similarity among different types of methyltransferases. This review aims to summarize the available information on the HhaI DNA methyltransferase.

[DNA-(N4-cytosine)-methyltransferase from Bacillus amyloliquefaciens: kinetic and substrate binding properties]. / DNK-(N4-tsitozin)-metiltransferaza iz Bacillus amyloliquefaciens: kineticheskie i substrat-sviazyvaiushchie svoistva.

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s-1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme-substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.

Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain.

The mouse (cytosine-5) DNA methyltransferase (Dnmt1) consists of a regulatory N-terminal and a catalytic C-terminal domain, which are fused by a stretch of Gly-Lys dipeptide repeats. The C-terminal region contains all of the conserved motifs found in other cytosine-5 DNA methyltransferases including the relative position of the catalytic Pro-Cys dipeptide. In prokaryotes, the methyltransferases are simpler and lack the regulatory N-terminal domain. We constructed three hybrid methyltransferases, containing the intact N-terminus of the murine Dnmt1 and most of the coding sequences from M.HhaI (GCGC), M.HpaII (CCGG) or M.SssI (CG). These hybrids are biologically active when expressed in a baculovirus system and show the specificity of the parental C-terminal domain. Expression of these recombinant constructs leads to de novo methylation of both host and viral genomes in a sequence-specific manner. Steady-state kinetic analyses were performed on the murine Dnmt1-HhaI hybrid using poly(dG-dC).poly (dG-dC), unmethylated and hemimethylated oligonucleotides as substrates. The enzyme has a slow catalytic turnover number of 4.38 h(-1) for poly(dG-dC). poly(dG-dC), and exhibits 3-fold higher catalytic efficiency for hemimethylated substrates.

The Ecl18kI restriction-modification system: cloning, expression, properties of the purified enzymes.

Ecl18kI is a type II restriction-modification system isolated from Enterobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransferase (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have been cloned and expressed in Escherichia coli. These enzymes recognize the 5'.../CCNGG...3' sequence in DNA; M.Ecl18kI methylates the C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as shown by the arrow. The restriction endonuclease and the methyltransferase were purified from E. coli B834 [p18Ap1] cells to near homogeneity. The restriction endonuclease is present in the solution as a tetramer, while the methyltransferase is a monomer. The interactions of M.Ecl18kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duplexes were investigated. The target base flipping-out mechanism is applicable in the case of M.Ecl18kI. Correct cleavage of the abasic substrates by R.Ecl18kI is accompanied by non-canonical hydrolysis of the modified strand.

Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific).

The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P2(1). Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.

Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides.

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103-108]. Here we describe protocols developed to purify polypeptides alpha and beta together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0 x 10(5) units per mg of protein. The respective denatured molecular masses of polypeptides alpha and beta were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 microM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase.

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.

Purification and characterization of a new DNA methyltransferase from Neisseria gonorrhoeae.

A new DNA methyltransferase, M.NgoBVII, was isolated from Neisseria gonorrhoeae strain WR302. M.NgoBVII recognizes the sequence 5'-GCNGC-3'.

M.BssHII: a new multispecific C5-DNA-methyltransferase.

M.BssHII is a new multispecific C5-DNA-methyltransferase recognizing five different targets. As the enzyme has been isolated from a thermophilic Bacillus, the protein should show enhanced intrinsic thermostability and therefore be a promising candidate for crystallizing a multispecific MTase.

In vitro DNA methylation by wheat nuclear cytosine DNA methyltransferase: effect of phytohormones.

Cytosine DNA methyltransferases (MTases) were isolated from nuclei of wheat seedlings and germinating embryos. The MTases isolated from both sources were able to perform de novo and maintenance DNA methylations. The most purified MTase fraction showed the presence of one main 67-kDa protein (embryos) and of a 85-kDa protein (in seedlings) in SDS-PAGE. Some plant growth regulators (gibberellic acid A3, 6-benzylaminopurine and fusicoccin) elevate by 30-65% the extent of in vitro DNA methylation by nuclear extracts with a maximal effect at 10(-6) M phytohormone concentration. The same phytohormones do not increase the extent of in vitro DNA methylation by purified wheat MTase; rather they inhibit it at concentrations of 10(-4)-10(-5) M. Thus, DNA methylation in the plant nucleus is controlled by phytohormones. The phytohormone effect may be mediated by other proteins in nuclear extracts.

The SsoII and NlaX DNA methyltransferases: overproduction and functional analysis.

Overproduction of the NlaX DNA methyltransferase (M.NlaX) in an Escherichia coli host conferred resistance to SsoII restriction endonuclease (R.SsoII) digestion. This suggested an overlap of sequence specificity between M.NlaX and M.SsoII, the latter of which modifies the internal cytosine of the target sequence 5'-CCNGG-3'. A variant of M.NlaX (M.Sso/Nla), containing an N-terminal extension from M.SsoII, was also enzymatically active. Using deletion analysis, the N-terminal 71 amino-acid residues of M.SsoII were shown to be essential for modification activity.

A bacterial methyltransferase M.EcoHK311 requires two proteins for in vitro methylation.

The genes encoding EcoHK311 restriction-modification (R-M) system were isolated from a clinically-isolated Escherichia coli strain HK31. The entire R-M system of EcoHK311 is located in a 2.1 kb fragment. R.EcoHK311 is an isoschizomer of Eael which recognizes and cleaves Y decreases GGCCR. M.EcoHK31l consists of two polypeptides alpha and beta with sizes 309 and 176 aa, respectively. Polypeptide beta is encoded within aa, alternative reading frame of polypeptide alpha. All the conserved motifs in mC5-MTases can be found in polypeptide alpha except motif IX which is present in polypeptide beta. Polypeptides alpha and beta were separately synthesized in a T7 promoter controlled over-expression system and in vitro methylation occurred only when the two extracts were mixed and thus confirms that two polypeptides are required for methylation.

The CpG-specific methylase SssI has topoisomerase activity in the presence of Mg2+.

A prokaryotic CpG-specific methylase from Spiroplasma, SssI methylase, is now widely used to study the effect of CpG methylation in mammalian cells, and can processively modify cytosines in CpG dinucleotides in the absence of Mg2+. In the presence of Mg2+, we found (i) that the methylation reaction is distributive rather than processive as a result of the decreased affinity of SssI methylase for DNA, and (ii) that a type I-like topoisomerase activity is present in SssI methylase preparations. This topoisomerase activity was still present in SssI methylase further purified by either SDS-polyacrylamide or isoelectric focusing gel electrophoresis. We show that methylase and topoisomerase activities are not functionally interdependent, since conditions exist where only one or the other enzymatic activity is detectable. The catalytic domains of SssI methylase and prokaryotic topoisomerases show similarity at the amino acid level, further supporting the idea that the topoisomerase activity is a genuine activity of SssI methylase. Mycoplasmas, including Spiroplasma, have the smallest genomes of all living organisms; thus, this condensation of two enzymatic activities into the same protein may be a result of genome economy, and may also have functional implications for the mechanism of methylation.

[New site site-specific endonuclease and methylase from Bacillus licheniformis 736]. / Novye sait-spetsificheskie éndonukleaza i metilaza iz Bacillus licheniformis 736.

The site-specific endonuclease R Bli736I and methylase M Bli736I have been isolated from the Bacillus licheniformis strain 736 by blue-agarose, hydroxyapatite-Ultragel and heparin-Sepharose chromatography. The enzymes are free from interfering impurities. R Bli736I recognizes the 5'-GGTCTCN-3' decreases and decreases 5'-NNNNNGAGACC-3' sequences on the DNA and cleaves the DNA as indicated by arrows to form single-stranded 4-nucleotide 5'-protruding termini. This enzyme is an isoschizomer of Eco3II isolated from E. coli.

Isolation and identification by sequence homology of a putative cytosine methyltransferase from Arabidopsis thaliana.

A plant cytosine methyltransferase cDNA was isolated using degenerate oligonucleotides, based on homology between prokaryote and mouse methyltransferases, and PCR to amplify a short fragment of a methyltransferase gene. A fragment of the predicted size was amplified from genomic DNA from Arabidopsis thaliana. Overlapping cDNA clones, some with homology to the PCR amplified fragment, were identified and sequenced. The assembled nucleic acid sequence is 4720 bp and encodes a protein of 1534 amino acids which has significant homology to prokaryote and mammalian cytosine methyltransferases. Like mammalian methylases, this enzyme has a C terminal methyltransferase domain linked to a second larger domain. The Arabidopsis methylase has eight of the ten conserved sequence motifs found in prokaryote cytosine-5 methyltransferases and shows 50% homology to the murine enzyme in the methyltransferase domain. The amino terminal domain is only 24% homologous to the murine enzyme and lacks the zinc binding region that has been found in methyltransferases from both mouse and man. In contrast to mouse where a single methyltransferase gene has been identified, a small multigene family with homology to the region amplified in PCR has been identified in Arabidopsis thaliana.

Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex.

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.