[Analysis of contacts between DNA and protein in a complex of SsoII mmethyltransferase-promoter region of the gene for the SsoII restriction-modification system].
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Amino Acid Sequence↗
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Results for “DNA Modification Methylases”
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ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation.
Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena. We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN (SUP) gene. The ago4-1 mutant reactivated silent SUP alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation. In addition, ago4-1 blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond to the retroelement AtSN1. These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG DNA methylation.
Alleles↗
[Expression of cloned gene for methyltransferase from Bacillus subtilis bacteriophage SPbetaB].
Expression of the methyltransferase gene from Bacillus subtilis lysogenizing phage SP beta B was studied by analyzing the sensitivity of the hybrid plasmid DNAs to restriction by the enzymes BspRI, Hpall and Mspl. The gene produces the methylase M. BsuP beta BI with specificity for 5'-GGCC. The fragment carrying the SP beta B derived gene also directs the synthesis in E. coli of a second methylase activity (M. BsuP beta BII) with 5'-CCGG specificity. Indirect evidence suggests that the two SP beta B modification activities are encoded by the same gene.
Bacillus subtilis↗
Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase.
The Eco RI endonuclease and methylase recognize the same hexanucleotide substrate sequence. We have determined the sequence of a fragment of DNA which encodes these enzymes using the chain-termination method of Sanger (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467). The amino acid sequences of both enzymes were derived from the DNA sequence. The coding regions selected include the only open translational frames of sufficient length to accommodate the enzymes. They coincide with previously established gene boundaries and orientation. The predicted amino acid sequences correlate well with analyses of the purified protein. Comparison of the nucleotide and protein sequences reveals no homology between the endonuclease and methylase which might provide insight into the origin of the restriction-modification system or the mechanism of common substrate recognition. Based on secondary structure predictions, the two enzymes also have grossly different molecular architecture. The base composition of the sequence is 65% A + T, and the codon usage is significantly different from that observed in several Escherichia coli chromosomal genes. In some cases, frequently selected codons are recognized by minor tRNA species. A spontaneous mutation in the endonuclease gene was isolated. Serine replaces arginine at residue 187. In crude extracts, Eco RI specific cleavage is approximately 0.3% wild type.
Amino Acid Sequence↗
Novel predicted RNA-binding domains associated with the translation machinery.
Two previously undetected domains were identified in a variety of RNA-binding proteins, particularly RNA-modifying enzymes, using methods for sequence profile analysis. A small domain consisting of 60-65 amino acid residues was detected in the ribosomal protein S4, two families of pseudouridine synthases, a novel family of predicted RNA methylases, a yeast protein containing a pseudouridine synthetase and a deaminase domain, bacterial tyrosyl-tRNA synthetases, and a number of uncharacterized, small proteins that may be involved in translation regulation. Another novel domain, designated PUA domain, after PseudoUridine synthase and Archaeosine transglycosylase, was detected in archaeal and eukaryotic pseudouridine synthases, archaeal archaeosine synthases, a family of predicted ATPases that may be involved in RNA modification, a family of predicted archaeal and bacterial rRNA methylases. Additionally, the PUA domain was detected in a family of eukaryotic proteins that also contain a domain homologous to the translation initiation factor eIF1/SUI1; these proteins may comprise a novel type of translation factors. Unexpectedly, the PUA domain was detected also in bacterial and yeast glutamate kinases; this is compatible with the demonstrated role of these enzymes in the regulation of the expression of other genes. We propose that the S4 domain and the PUA domain bind RNA molecules with complex folded structures, adding to the growing collection of nucleic acid-binding domains associated with DNA and RNA modification enzymes. The evolution of the translation machinery components containing the S4, PUA, and SUI1 domains must have included several events of lateral gene transfer and gene loss as well as lineage-specific domain fusions.
Amino Acid Sequence↗
Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein.
The DdeI restriction-modification system was previously cloned and has been maintained in E. coli on two separate and compatible plasmids (1). The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively. Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning. The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E. coli. Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322. The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro. Finally, comparison of the deduced amino acid sequence of M. DdeI with that of other known cytosine methylases shows significant regions of homology.
Amino Acid Sequence↗
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.
Amino Acid Sequence↗
Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis.
DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.
Adenine↗
Cytosine methylation determines hot spots of DNA damage in the human P53 gene.
In the P53 tumor suppressor gene, a remarkably large number of somatic mutations are found at methylated CpG dinucleotides. We have previously mapped the distribution of (+/-) anti-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy -7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) adducts along the human P53 gene [Denissenko, M. F., Pao, A., Tang, M.-s. & Pfeifer, G. P. (1996) Science 274, 430-432]. Strong and selective formation of adducts occurred at guanines in CpG sequences of codons 157, 248, and 273, which are the major mutational hot spots in lung cancer. Chromatin structure was not involved in preferential modification of these sites by BPDE. To investigate other possible mechanisms underlying the selectivity of BPDE binding, we have mapped the adducts in plasmid DNA containing genomic P53 sequences. The adduct profile obtained was different from that in genomic DNA. However, when cytosines at CpG sequences were converted to 5-methylcytosines by the CpG-specific methylase SssI and the DNA was subsequently treated with BPDE, adduct hot spots were created which were similar to those seen in genomic DNA where all CpGs are methylated. A strong positive effect of 5-methylcytosine on BPDE adduct formation at CpG sites was also documented with sequences of the PGK1 gene derived from an active or inactive human X chromosome and having differential methylation patterns. These results show that methylated CpG dinucleotides, in addition to being an endogenous promutagenic factor, may represent a preferential target for exogenous chemical carcinogens. The data open new avenues concerning the reasons that the majority of mutational hot spots in human genes are at CpGs.
Base Sequence↗
Esp1396I restriction-modification system: structural organization and mode of regulation.
Esp1396I restriction-modification (RM) system recognizes an interrupted palindromic DNA sequence 5'-CCA(N)(5)TGG-3'. The Esp1396I RM system was found to reside on pEsp1396, a 5.6 kb plasmid naturally occurring in Enterobacter sp. strain RFL1396. The nucleotide sequence of the entire 5622 bp pEsp1396 plasmid was determined on both strands. Identified genes for DNA methyltransferase (esp1396IM) and restriction endonuclease (esp1396IR) are transcribed convergently. The restriction endonuclease gene is preceded by the small ORF (esp1396IC) that possesses a strong helix-turn-helix motif and resembles regulatory proteins found in PvuII, BamHI and few other RM systems. Gene regulation studies revealed that C.Esp1396I acts as both a repressor of methylase expression and an activator of regulatory protein and restriction endonuclease expression. Our data indicate that C protein from Esp1396I RM system activates the expression of the Enase gene, which is co-transcribed from the promoter of regulatory gene, by the mechanism of coupled translation.
Base Sequence↗
Selection of mutations altering specificity in restriction-modification enzymes using the bacteriophage P22 challenge-phage system.
A method for selecting mutants of site-specific DNA-binding proteins has been applied to the study of the EcoRI and RsrI restriction-modification enzymes. Catalytically inactive variants of both endonucleases are shown to function as pseudo-repressors in the bacteriophage P22 challenge-phage assay, and, upon further mutagenesis of the gene encoding R.EcoRI, a variant of that enzyme has been selected which appears to bind EcoRI-methylated GAATTC sequences to the exclusion of unmethylated sites: this specificity is the opposite of that belonging to the native enzyme. Variants of the EcoRI methylase have also been found that lack either catalytic activity or both binding and catalytic activities.
Bacteriophage P22↗
Probing the role of cysteine residues in the EcoP15I DNA methyltransferase.
Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.
Amino Acid Substitution↗
Cloning, over-expression and the catalytic properties of the EcoP15 modification methylase from Escherichia coli.
The EcoP15 modification methylase gene from the p15B plasmid of Escherichia coli 15T-has been cloned and expressed at high levels in a plasmid vector system. We have purified the enzyme to near homogeneity in large amounts and have studied some of its enzymatic properties. Initial rates of methyl transfer are first order in methylase concentration and, with pUC19 DNA as substrate, the reaction proceeds by a random mechanism in which either DNA or S-adenosylmethionine can bind to the free enzyme. After methyltransfer to DNA, the methylated DNA and S-adenosylhomocysteine appear to dissociate in random order. As expected in such a mechanism, S-adenosylhomocysteine is a non-competitive inhibitor by S-adenosylmethionine at concentrations not much above its KM suggests that release of methylated DNA may be the rate-limiting step. This suggestion is strengthened by the fact that a mutant of the closely related EcoP1 does not show such substrate inhibition.
Binding, Competitive↗
Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli.
A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized. This plasmid, pLB1 , is 6.2 kb long and carries only the Eco RV genes. A subclone of 3 kb has been inserted in pBR322. The relative positions of the endonuclease and the methylase genes were determined by the construction of a set of overlapping deletions generated by Bal31 resection. The DNA sequence of a 2.2 kb fragment containing the two genes was determined. The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis. Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed.
Amino Acid Sequence↗
Cytosine methylation enhances DNA damage produced by groove binding and intercalating enediynes: studies with esperamicins A1 and C.
Methylation of the C5 position of cytosine in CG dinucleotides represents an important element in the control of gene expression in eukaryotic cells. This major groove modification of DNA causes changes in DNA conformation that are recognized by DNA-binding proteins and DNA-damaging chemicals. We have observed that CG methylation affects the DNA damage produced by the enediyne esperamicin A1 and its analog lacking the intercalating anthranilate, esperamicin C. Fragments of the human phosphoglycerate kinase gene (PGK1) and the plasmid pUC19 were methylated with SssI methylase and subjected to damage by esperamicins A1 and C. Damage produced by esperamicin A1 was enhanced 1.5-2-fold near a single CG sequence at position -101 in PGK1 and in a region containing several methylated CG dinucleotides between positions -120 and -131. Esperamicin C-induced damage was enhanced to a similar degree in PGK1 but only at the site that contained multiple CG dinucleotides. There was enhancement of damage for both drugs in the pUC19 fragment at several sites near CG sequences. Analysis of the chemistry of esperamicin-induced DNA damage suggests that cytosine methylation does not affect the identity of drug-abstracted hydrogen atoms. The damage chemistry was also used to identify the DNA binding orientation of the esperamicins. The anthranilate of esperamicin A1 is predicted to intercalate in a CT step four base pairs in a 3'-direction to the CG sequence at -101 in PGK1 and in a CG dinucleotide at the site containing multiple CGs (positions -120 to -131). These observations are consistent with other studies that indicate a long range effect of cytosine methylation on DNA conformation.
5-Methylcytosine↗
Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.
To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.
Bacterial Proteins↗
[The effect of point modifications of sugar-phosphate backbone of DNA on function of EcoRII, MvaI, and BstN1 restriction endonucleases].
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Base Sequence↗
NmeSI restriction-modification system identified by representational difference analysis of a hypervirulent Neisseria meningitidis strain.
Neisseria meningitidis is a gram-negative bacterium that may cause meningitis, sepsis, or both. The increase in the incidence of meningococcal disease in various countries in the past 2 decades is mainly due the genotypically related lineage III meningococci. The chromosomal DNA differences between lineage III strains and non-lineage III strains were identified using representational difference analysis. Thus, a 1.8-kb locus that is specific for lineage III meningococci was identified. The locus contains three open reading frames encoding the NmeSI restriction-modification system. The methyltransferase gene was cloned and expressed in Escherichia coli. Site AGTACT was found to be modified by the enzyme. In conclusion, lineage III strains differ from endemic strains by the presence of a specific restriction-modification system. This restriction-modification system may contribute to the clonal and hypervirulent character of lineage III strains by influencing horizontal gene transfer and transcription.
Amino Acid Sequence↗