Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.     

The Epigenomic Landscape of Prokaryotes

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.     

DsaV methyltransferase and its isoschizomers contain a conserved segment that is similar to the segment in Hhai methyltransferase that is in contact with DNA bases.

The methyltransferase (MTase) in the DsaV restriction--modification system methylates within 5'-CCNGG sequences. We have cloned the gene for this MTase and determined its sequence. The predicted sequence of the MTase protein contains sequence motifs conserved among all cytosine-5 MTases and is most similar to other MTases that methylate CCNGG sequences, namely M.ScrFI and M.SsoII. All three MTases methylate the internal cytosine within their recognition sequence. The 'variable' region within the three enzymes that methylate CCNGG can be aligned with the sequences of two enzymes that methylate CCWGG sequences. Remarkably, two segments within this region contain significant similarity with the region of M.HhaI that is known to contact DNA bases. These alignments suggest that many cytosine-5 MTases are likely to interact with DNA using a similar structural framework.     

Is the HemK family of putative S-adenosylmethionine-dependent methyltransferases a "missing" zeta subfamily of adenine methyltransferases? A hypothesis

Previous comparative studies revealed close similarity among various groups of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases), indicating their common evolutionary origin. We present evidence for a remarkable similarity between the sequence and predicted structure of HemK (a widespread family of putative proteins encoded in genomes from bacteria to humans) and the catalytic domain of the gamma-subfamily of adenine-specific DNA MTases (N6mA MTases). We predict the structure and function of the putative catalytic domain of HemK proteins and speculate that the target-recognizing function may be conferred by the N-terminal variable region.     

鳜鱼传染性脾肾坏死病毒的胞嘧啶5-甲基转移酶基因结构及其序列分析

对鳜鱼传染性脾肾坏死病毒（infectious spleen and kidney necrosis virus,ISKNV）的胞嘧啶5－甲基转移酶（MTase）基因的结构及序列进行了分析。序列比较分析表明,ISKNV MTase编码区全长684bp,编码长227个氨基酸的蛋白质,推测分子量为25855D。与一些细菌的MTase比较,ISKNV MTase也含有负责转移甲基的4个保守区,但缺乏识别靶序列的保守区。比较ISKNV与其它6种脊椎动物虹彩病毒的MTase序列并建立系统树,ISKNV显著不同于蛙病毒属和淋巴囊肿病毒属。7种脊椎动物虹彩病毒MTase具有高度保守区,可以此设计引物用PCR方法鉴定脊椎动物虹彩病毒。     

DNA binding properties in vivo and target recognition domain sequence alignment analyses of wild-type and mutant RsrI [N6-adenine] DNA methyltransferases

A genetic selection method, the P22 challenge-phage assay, was used to characterize DNA binding in vivo by the prokaryotic beta class [N:6-adenine] DNA methyltransferase M.RSR:I. M.RSR:I mutants with altered binding affinities in vivo were isolated. Unlike the wild-type enzyme, a catalytically compromised mutant, M.RSR:I (L72P), demonstrated site-specific DNA binding in vivo. The L72P mutation is located near the highly conserved catalytic motif IV, DPPY (residues 65-68). A double mutant, M.RSR:I (L72P/D173A), showed less binding in vivo than did M.RSR:I (L72P). Thus, introduction of the D173A mutation deleteriously affected DNA binding. D173 is located in the putative target recognition domain (TRD) of the enzyme. Sequence alignment analyses of several beta class MTases revealed a TRD sequence element that contains the D173 residue. Phylogenetic analysis suggested that divergence in the amino acid sequences of these methyltransferases correlated with differences in their DNA target recognition sequences. Furthermore, MTases of other classes (alpha and gamma) having the same DNA recognition sequence as the beta class MTases share related regions of amino acid sequences in their TRDs.     

Structure, function and mechanism of exocyclic DNA methyltransferases

DNA MTases (methyltransferases) catalyse the transfer of methyl groups to DNA from AdoMet (S-adenosyl-L-methionine) producing AdoHcy (S-adenosyl-L-homocysteine) and methylated DNA. The C5 and N4 positions of cytosine and N6 position of adenine are the target sites for methylation. All three methylation patterns are found in prokaryotes, whereas cytosine at the C5 position is the only methylation reaction that is known to occur in eukaryotes. In general, MTases are two-domain proteins comprising one large and one small domain with the DNA-binding cleft located at the domain interface. The striking feature of all the structurally characterized DNA MTases is that they share a common core structure referred to as an 'AdoMet-dependent MTase fold'. DNA methylation has been reported to be essential for bacterial virulence, and it has been suggested that DNA adenine MTases (Dams) could be potential targets for both vaccines and antimicrobials. Drugs that block Dam could slow down bacterial growth and therefore drug-design initiatives could result in a whole new generation of antibiotics. The transfer of larger chemical entities in a MTase-catalysed reaction has been reported and this represents an interesting challenge for bio-organic chemists. In general, amino MTases could therefore be used as delivery systems for fluorescent or other reporter groups on to DNA. This is one of the potential applications of DNA MTases towards developing non-radioactive DNA probes and these could have interesting applications in molecular biology. Being nucleotide-sequence-specific, DNA MTases provide excellent model systems for studies on protein-DNA interactions. The focus of this review is on the chemistry, enzymology and structural aspects of exocyclic amino MTases.     

Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ

RlmJ catalyzes the m⁶A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence ₁₆₄DPPY₁₆₇ is more similar to DNA m⁶A MTases than to RNA m⁶₂A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.     

Identification of a subdomain within DNA-(cytosine-C5)-methyltransferases responsible for the recognition of the 5' part of their DNA target.

In previous work on DNA-(cytosine-C5)-methyltransferases (C5-MTases), domains had been identified which are responsible for the sequence specificity of the different enzymes (target-recognizing domains, TRDs). Here we have analyzed the DNA methylation patterns of two C5-MTases containing reciprocal chimeric TRDs, consisting of the N- and C-terminal parts derived from two different parental TRDs specifying the recognition of 5'-CC(A/T)GG-3' and 5'-GCNGC-3'. Sequences recognized by these engineered MTases were non-symmetrical and degenerate, but contained at their 5' part a consensus sequence which was very similar to the 5' part of the target recognized by the parental TRD which contributed the N-terminal moiety of the chimeric TRD. The results are discussed in connection with the present understanding of the mechanism of DNA target recognition by C5-MTases. They demonstrate the possibility of designing C5-MTases with novel DNA methylation specificities.     

Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars

The methylation of DNA bases plays an important role in numerous biological processes including development, gene expression, and DNA replication. Salmonella is an important foodborne pathogen, and methylation in Salmonella is implicated in virulence. Using single molecule real-time (SMRT) DNA-sequencing, we sequenced and assembled the complete genomes of eleven Salmonella enterica isolates from nine different serovars, and analysed the whole-genome methylation patterns of each genome. We describe 16 distinct N⁶-methyladenine (m6A) methylated motifs, one N⁴-methylcytosine (m4C) motif, and one combined m6A-m4C motif. Eight of these motifs are novel, i.e., they have not been previously described. We also identified the methyltransferases (MTases) associated with 13 of the motifs. Some motifs are conserved across all Salmonella serovars tested, while others were found only in a subset of serovars. Eight of the nine serovars contained a unique methylated motif that was not found in any other serovar (most of these motifs were part of Type I restriction modification systems), indicating the high diversity of methylation patterns present in Salmonella.     

Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo

While CpG methylation can be readily analyzed at the DNA sequence level in wild-type and mutant cells, the actual DNA (cytosine-5) methyltransferases (DNMTs) responsible for in vivo methylation on genomic DNA are less tractable. We used an antibody-based method to identify specific endogenous DNMTs (DNMT1, DNMT1b, DNMT2, DNMT3a, and DNMT3b) that stably and selectively bind to genomic DNA containing 5-aza-2'-deoxycytidine (aza-dC) in vivo. Selective binding to aza-dC-containing DNA suggests that the engaged DNMT is catalytically active in the cell. DNMT1b is a splice variant of the predominant maintenance activity DNMT1, while DNMT2 is a well-conserved protein with homologs in plants, yeast, Drosophila, humans, and mice. Despite the presence of motifs essential for transmethylation activity, catalytic activity of DNMT2 has never been reported. The data here suggest that DNMT2 is active in vivo when the endogenous genome is the target, both in human and mouse cell lines. We quantified relative global genomic activity of DNMT1, -2, -3a, and -3b in a mouse teratocarcinoma cell line. DNMT1 and -3b displayed the greatest in vivo binding avidity for aza-dC-containing genomic DNA in these cells. This study demonstrates that individual DNMTs can be tracked and that their binding to genomic DNA can be quantified in mammalian cells in vivo. The different DNMTs display a wide spectrum of genomic DNA-directed activity. The use of an antibody-based tracking method will allow specific DNMTs and their DNA targets to be recovered and analyzed in a physiological setting in chromatin.     

Characterization of the large subunit of EcoHK31I methyltransferase by structural modeling and mutagenesis

M.EcoHK31I is a naturally occurring mC5-methyltransferase with a large alpha polypeptide and a small beta polypeptide. Polypeptide alpha contains conserved motifs I-VIII and X, and polypeptide beta contains motif IX. To understand how polypeptide alpha carries out its function, a molecular model of the large domain of polypeptide alpha was generated using M.HhaI and M.HaeIII as templates. The large domain is a mixed alpha/beta structure. Residues 15-19 in motif I (Phe-Naa-Gly-Naa) are conserved for cofactor binding. The key catalytic residue Cys-79 in motif IV is also conserved in comparison with other C-5 MTases. Comparing polypeptide alpha with M.HhaI and M.HaeIII revealed a unique region upstream of motif X. To understand the role of this region, 14 charged residues between R224 and E271 in the putative small domain were mutated. Activity assays indicated that most of these charges can be eliminated or changed conservatively. Among these charged residues, R224, E240, D245 and D251 may take part in proper interaction with DNA in the presence of polypeptide beta.     

Cloning of CviPII nicking and modification system from chlorella virus NYs-1 and application of Nt.CviPII in random DNA amplification

The cloning and expression of the CviPII DNA nicking and modification system encoded by chlorella virus NYs-1 is described. The system consists of a co-linear MTase encoding gene (cviPIIM) and a nicking endonuclease encoding gene (cviPIINt) separated by 12 nt. M.CviPII possesses eight conserved amino acid motifs (I to VIII) typical of C5 MTases, but, like another chlorella virus MTase M.CviJI, lacks conserved motifs IX and X. In addition to modification of the first cytosine in CCD (D = A, G or T) sequences, M.CviPII modifies both the first two cytosines in CCAA and CCCG sites as well. Nt.CviPII has significant amino acid sequence similarity to Type II restriction endonuclease CviJI that recognizes an overlapping sequence (RG--CY). Nt.CviPII was expressed in Escherichia coli with or without a His-tag in a host pre-modified by M.CviPII. Recombinant Nt.CviPII recognizes the DNA sequence CCD and cleaves the phosphodiester bond 5' of the first cytosine while the other strand of DNA at this site is not affected. Nt.CviPII displays site preferences with CCR (R = A or G) sites preferred over CCT sites. Nt.CviPII is active from 16 to 65 degrees C with a temperature optimum of 30-45 degrees C. Nt.CviPII can be used to generate single-stranded DNAs (ssDNAs) for isothermal strand-displacement amplification. Nt.CviPII was used in combination with Bst DNA polymerase I large fragment to rapidly amplify anonymous DNA from genomic DNA or from a single bacterial colony.     

Recombinant human DNA (cytosine-5) methyltransferase. II. Steady-state kinetics reveal allosteric activation by methylated dna.

Initial velocity determinations were conducted with human DNA (cytosine-5) methyltransferase (DNMT1) on unmethylated and hemimethylated DNA templates in order to assess the mechanism of the reaction. Initial velocity data with DNA and S-adenosylmethionine (AdoMet) as variable substrates and product inhibition studies with methylated DNA and S-adenosylhomocysteine (AdoHcy) were obtained and evaluated as double-reciprocal plots. These relationships were linear for plasmid DNA, exon-1 from the imprinted small nuclear ribonucleoprotein-associated polypeptide N, (CGG.CCG)(12), (m(5)CGG. CCG)(12), and (CGG.CCG)(73) but were not linear for (CGG. Cm(5)CG)(12). Inhibition by AdoHcy was apparently competitive versus AdoMet and uncompetitive/noncompetitive versus DNA at 相似文献   

Human DNMT2 methylates tRNA(Asp) molecules using a DNA methyltransferase-like catalytic mechanism

Although their amino acid sequences and structure closely resemble DNA methyltransferases, Dnmt2 proteins were recently shown by Goll and colleagues to function as RNA methyltransferases transferring a methyl group to the C5 position of C38 in tRNA(Asp). We observe that human DNMT2 methylates tRNA isolated from Dnmt2 knock-out Drosophila melanogaster and Dictyostelium discoideum. RNA extracted from wild type D. melanogaster was methylated to a lower degree, but in the case of Dictyostelium, there was no difference in the methylation of RNA isolated from wild-type and Dnmt2 knock-out strains. Methylation of in vitro transcribed tRNA(Asp) confirms it to be a target of DNMT2. Using site directed mutagenesis, we show here that the enzyme has a DNA methyltransferase-like mechanism, because similar residues from motifs IV, VI, and VIII are involved in catalysis as identified in DNA methyltransferases. In addition, exchange of C292, which is located in a CFT motif conserved among Dnmt2 proteins, strongly reduced the catalytic activity of DNMT2. Dnmt2 represents the first example of an RNA methyltransferase using a DNA methyltransferase type of mechanism.     

Sequence motifs specific for cytosine methyltransferases

Using a new alignment method, the sequences of 13 m5C methyltransferases (MTases) have been examined. Five extremely well-conserved blocks of sequence have been detected and have been used as fixed points for the alignment of the 13 sequences. Following this initial alignment, five further blocks of similarity have been identified to give a total of ten recognizable blocks of sequence homology that are all arranged in a common order. The structures of these MTases consist of a variable-length N-terminal arm followed by eight well-conserved blocks each separated by small variable-length regions. A large variable-length segment of 90 to 270 amino acids (aa) then follows. After this are two blocks, and a variable-length C-terminal segment completes the sequence. Within the final alignment, 20 aa in the protein sequences, and 86 nucleotides in the nucleotide sequences are invariant. The strongest conservation is found in proximity to a suspected functional site that contains the dipeptide proline-cysteine. Consensus patterns can be defined for the five best conserved blocks and, when used as search motifs, are able to clearly distinguish between the m5C MTases and all other identified proteins in the PIR database. This suggests they may be of use in identifying putative MTases among protein sequences of unknown function.     

The eukaryotic DNMT2 genes encode a new class of cytosine-5 DNA methyltransferases

DNMT2 is a subgroup of the eukaryotic cytosine-5 DNA methyltransferase gene family. Unlike the other family members, proteins encoded by DNMT2 genes were not known before to possess DNA methyltransferase activities. Most recently, we have shown that the genome of Drosophila S2 cells stably expressing an exogenous Drosophila dDNMT2 cDNA became anomalously methylated at the 5'-positions of cytosines (Reddy, M. N., Tang, L. Y., Lee, T. L., and Shen, C.-K. J. (2003) Oncogene, in press). We present evidence here that the genomes of transgenic flies overexpressing the dDnmt2 protein also became hypermethylated at specific regions. Furthermore, transient transfection studies in combination with sodium bisulfite sequencing demonstrated that dDnmt2 as well as its mouse ortholog, mDnmt2, are capable of methylating a cotransfected plasmid DNA. These data provide solid evidence that the fly and mouse DNMT2 gene products are genuine cytosine-5 DNA methyltransferases.     

Diverse DNA modification in marine prokaryotic and viral communities

DNA chemical modifications, including methylation, are widespread and play important roles in prokaryotes and viruses. However, current knowledge of these modification systems is severely biased towards a limited number of culturable prokaryotes, despite the fact that a vast majority of microorganisms have not yet been cultured. Here, using single-molecule real-time sequencing, we conducted culture-independent ‘metaepigenomic’ analyses (an integrated analysis of metagenomics and epigenomics) of marine microbial communities. A total of 233 and 163 metagenomic-assembled genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTase genes were not genetically linked with the endonuclease genes predicted to be involved in defense mechanisms against extracellular DNA. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, 5 of which showed novel specificities and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative specificities in MTases that are highly conserved in Alphaproteobacteria, which may enhance our understanding of the co-evolutionary history of the methylation systems and the genomes. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses in nature.     

Exact size and organization of DNA target-recognizing domains of multispecific DNA-(cytosine-C5)-methyltransferases.

A large portion of the sequences of type II DNA-(cytosine-C5)-methyltransferases (C5-MTases) represent highly conserved blocks of amino acids. General steps in the methylation reaction performed by C5-MTases have been found to be mediated by some of these domains. C5-MTases carry, in addition at the same relative location, a region variable in size and amino acid composition, part of which is associated with the capacity of each C5-MTase to recognize its characteristic target. Individual target-recognizing domains (TRDs) for the targets CCGG (M), CC(A/T)GG (E), GGCC (H), GCNGC (F) and G(G/A/T)GC(C/A/T)C (B) could be identified in the C-terminal part of the variable region of multispecific C5-MTases. With experiments reported here, we have established the organization of the variable regions of the multispecific MTases M.SPRI, M.phi3TI, M.H2I and M.rho 11SI at the resolution of individual amino acids. These regions comprise 204, 175, 268 and 268 amino acids, respectively. All variable regions are bipartite. They contain at their N-terminal side a very similar sequence of 71 amino acids. The integrity of this sequence must be assured to provide enzyme activity. Bracketed by 6-10 'linker' amino acids, they have, depending on the enzyme studied, towards their C-terminal end ensembles of individual TRDs of 38 (M), 39 (E), 40 (H), 44 (F) and 54 (B) amino acids. TRDs of different enzymes with equal specificity have the same size. TRDs do not overlap but are either separated by linker amino acids or abut each other.