Molecular cloning, sequencing, and mapping of the bacteriophage T2 dam gene.

Bacteriophage T2 codes for a DNA-(adenine-N6)methyltransferase (Dam), which is able to methylate both cytosine- and hydroxymethylcytosine-containing DNAs to a greater extent than the corresponding methyltransferase encoded by bacteriophage T4. We have cloned and sequenced the T2 dam gene and compared it with the T4 dam gene. In the Dam coding region, there are 22 nucleotide differences, 4 of which result in three coding differences (2 are in the same codon). Two of the amino acid alterations are located in a region of homology that is shared by T2 and T4 Dam, Escherichia coli Dam, and the modification enzyme of Streptococcus pneumoniae, all of which methylate the sequence 5' GATC 3'. The T2 dam and T4 dam promoters are not identical and appear to have slightly different efficiencies; when fused to the E. coli lacZ gene, the T4 promoter produces about twofold more beta-galactosidase activity than does the T2 promoter. In our first attempt to isolate T2 dam, a truncated gene was cloned on a 1.67-kilobase XbaI fragment. This construct produces a chimeric protein composed of the first 163 amino acids of T2 Dam followed by 83 amino acids coded by the pUC18 vector. Surprisingly, the chimera has Dam activity, but only on cytosine-containing DNA. Genetic and physical analyses place the T2 dam gene at the same respective map location as the T4 dam gene. However, relative to T4, T2 contains an insertion of 536 base pairs 5' to the dam gene. Southern blot hybridization and computer analysis failed to reveal any homology between this insert and either T4 or E. coli DNA.     

The DNA and S-adenosylmethionine-binding regions of EcoDam and related methyltransferases

Previous comparison of the amino acid sequences of the GATC-methylating Escherichia coli Dam methyltransferase (MTase) with those of other adenine MTases (M.EcoRV, M.DpnII and T4Dam) localized four conserved regions. Regions III and IV have similarities with many other MTases. The sequence DPPY (or NPPY) is always present in region IV. It was suggested to be the AdoMet binding site. Publication of the nucleotide and amino acid sequences of M.CviBIII, M.DpnA and MutH give further credence to this assignment: M.DpnA, which also methylates GATC, has strong similarities with regions III and IV; M.CviBIII, a cytosine methylase, has a characteristic NPPY sequence in region IV, and only limited resemblance in region III; MutH, the GATC-specific endonuclease in DNA mismatch repair, has significant similarities uniquely in region III. The presently available evidence suggests that region III is the GAT(C) binding site and region IV is the AdoMet binding site. This hypothesis is strengthened by recent genetic findings.     

The bacteriophage T2 and T4 DNA-[N6-adenine] methyltransferase (Dam) sequence specificities are not identical.

Bacteriophages T2 and T4 encode DNA-[N6-adenine] methyltransferases (Dam) which differ from each other by only three amino acids. The canonical recognition sequence for these enzymes in both cytosine and 5-hydroxymethylcytosine-containing DNA is GATC; at a lower efficiency they also recognize some non-canonical sites in sequences derived from GAY (where Y is cytosine or thymine). We found that T4 Dam fails to methylate certain GATA and GATT sequences which are methylated by T2 Dam. This indicates that T2 Dam and T4 Dam do not have identical sequence specificities. We analyzed DNA sequence data files obtained from GenBank, containing about 30% of the T4 genome, to estimate the overall frequency of occurrence of GATC, as well as non-canonical sites derived from GAY. The observed N6methyladenine (m6A) content of T4 DNA, methylated exclusively at GATC (by Escherichia coli Dam), was found to be in good agreement with this estimate. Although GATC is fully methylated in virion DNA, only a small percentage of the non-canonical sequences are methylated.     

Characterization of a dam Mutant of Serratia marcescens and Nucleotide Sequence of the dam Region

The DNA of Serratia marcescens has N6-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from S. marcescens Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (dam) coding for the adenine methyltransferase (Dam enzyme) of S. marcescens was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a dam-13::Tn9 mutant of Escherichia coli. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of E. coli. The dam gene is located between flanking genes which are similar to those found to the sides of the E. coli dam gene. The results of complementation studies indicated that like Dam of E. coli and unlike Dam of Vibrio cholerae, the Dam enzyme of S. marcescens plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors.     

Methylation by a mutant T2 DNA [N(6)-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage

Properties of a mutant bacteriophage T2 DNA [N:(6)-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher k(cat) in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage lambda DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BCL:I and ECO:RV endonucleases has been shown on phage lambda DNA and with BCL:I and DPN:II endonucleases on yeast chromosomal DNA embedded in agarose.     

Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.

The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.     

Molecular enzymology of phage T4 Dam DNA-methyltransferase

The review reflects results of studies on the molecular mechanism of phage T4 Dam DNA-methyltransferase action. The enzyme (T4Dam) catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to N6-adenine position in the palindromic recognition sequence GATC (EC 2.1.1.72). The enzyme subunit structure, substrate-binding and kinetic parameters for a wide range of native and modified oligonucleotide duplexes, as well as steady-state reaction kinetic scheme, included T4Dam isomerization to catalytically active form, are considered. The found mechanisms of DNA induced T4Dam dimerization, target base flipping, enzyme reorientation in an asymmetrically modified recognition sequence, effector action of reaction substrates and processive methylation of DNA substrates, containing more than one specific site, are discussed. The results obtained with T4Dam may be useful for understanding mechanisms of action of other homologous enzymes, most of all for specimens of numerous family of Dam DNA-methyltransferases.     

Identification and characterization of a Treponema pallidum subsp. pallidum gene encoding a DNA adenine methyltransferase

The nucleotide sequence of a DNA adenine methyltransferase gene (dam) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The dam gene encodes a 303 amino acid protein whose deduced sequence has significant homology with DNA (N⁶-adenine) methyltransferases. T. pallidum Dam can be assigned to group α DNA amino methyltransferases based on the order of nine conserved motifs that are present in the protein. Digests of T. pallidum chromosomal DNA performed with isoschizomer restriction endonucleases (Sau3AI, DpnI, and MboI) confirmed the presence of methylated adenine residues in GATC sequences (Dam⁺ phenotype).     

Differential methylation kinetics of individual target site strands by T4Dam DNA methyltransferase

Prokaryote DNA methyltransferases (MTases) of the Dam family (including those of bacteriophages T2 and T4) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. Dam DNA MTases, as all site-specific enzymes interacting with polymeric DNA, require a mechanism of action that ensures a rapid search for specific targets for catalytic action, during both the initial and subsequent rounds of methylation. The results of pre-steady-state (reaction burst) and steady-state methylation analyses of individual targets permitted us to monitor the action of T4Dam, which has three degrees of freedom: sliding, reorientation and adaptation to the canonical GATC sequence. The salient results are as follows: (i) 40mer substrate duplexes containing two canonical GATC sites showed differential methylation of the potential targets, i.e., T4Dam exhibited a preference for one site/target, which may present the better 'kinetic trap' for the enzyme. (ii) Prior hemimethylation of the two sites made both targets equally capable of being methylated during the pre-steady-state reaction. (iii) Although capable of moving in either direction along double-stranded DNA, there are some restrictions on T4Dam reorientation/adaptation on 40mer duplexes.     

Substitution of a single bacteriophage T3 residue in bacteriophage T7 RNA polymerase at position 748 results in a switch in promoter specificity.

The bacteriophage T3 and T7 RNA polymerases (RNAP) are closely related, yet exhibit high specificity for their own promoter sequences. In this work the primary determinant of T7 versus T3 promoter specificity has been localized to a single amino acid residue at position 748 in the T7 RNAP. Substitution of this residue (Asn) with the corresponding residue found in T3 RNAP (Asp) results in a switch in promoter specificity, and specifically alters recognition of the base pairs (bp) at positions -11 and, possibly, -10 in the promoter. A complementary mutation in T3 RNAP (T3-D749N) results in a similar switch in promoter preference for that enzyme. The hierarchy of bp preference by the mutant and wild-type enzymes for bp at -10 and -11, and the results of previous experiments, lead to a model for specificity in which it is proposed that N748 in T7 RNAP (and D749 in T3 RNAP) make specific hydrogen bonds with bases at -11 and -10 on the non-template strand in the major groove. The specificity determining region of T7 RNAP does not appear to exhibit homology to any known sequence-dependent DNA binding motif.     

Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1

The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.     

Structure of the bacteriophage T4 DNA adenine methyltransferase

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket.

Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the beta-1,3-1,4-glucanase gene of Bacillus macerans: homologies to other beta-glucanases

Summary The nucleotide sequence of an 852 base pair (bp) DNA fragment containing the entire gene coding for thermostable beta- 1,3-1,4-glucanase ofBacillus macerans has been determined. ThebglM gene comprises an open reading frame (ORF) of 711 by (237 codons) starting with ATG at position 93 and extending to the translational stop codon TAA at position 804. The deduced amino acid sequence of the mature protein shows 70% homology to published sequences of mesophilic beta- 1,3-1,4-glucanases fromB. subtilis andB. amyloliquefaciens. The sequence coding for mature beta-glucanase is preceded by a putative signal peptide of 25 amino acid residues, and a sequence resembling a ribosome-binding site (GGAGG) before the initiation codon. By contrast with the processed protein, the N-terminal amino acid sequence constituting the putative leader peptide bears no or only weak homology to signal peptides of mesophilicBacillus endo-beta-glucanases. TheB. macerans signal peptide appears to be functional in exporting the enzyme to the periplasm inE. coli. More than 50% of the whole glucanase activity was localized in the periplasmic space and in the supernatant. Whereas homology to endo-1,4-beta-glucanases is completely lacking, a weak amino acid homology between the sequence surrounding the active site of phage T4 lysozyme and a sequence spanning residues 126 through 161 ofB. macerans endo-beta-glucanase could be identified.     

Molecular cloning of the nahG gene encoding salicylate hydroxylase from Pseudomonas fluorescens

A gene encoding the salicylate hydroxylase was cloned from the genomic DNA of Pseudomonas fluorescens SME11. The DNA fragment containing the nahG gene for the salicylate hydroxylase was mapped with restriction endonucleases and sequenced. The DNA fragment contained an ORF of 1,305 bp encoding a polypeptide of 434 amino acid residues. The nucleotide and amino acid sequences of the salicylate hydroxylase revealed several conserved regions with those of the enzyme encoded in P. putida PpG7: The homology of the nucleotide sequence is 83% and that of amino acid sequence is 72%. We found large conserved regions of the amino acid sequence at FAD and NADH binding regions. The FAD binding site is located at the amino terminal region and a lysine residue functions as a NADH-binding site.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8.

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.     

Symmetry elements in DNA structure important for recognition/methylation by DNA [amino]-methyltransferases

The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in k_cat was observed for all three enzymes if a particular element of structural symmetry was disrupted. For the BamHI MTase, integrity of the ATCC was critical, while an intact GAT sequence was necessary for the activity of T4Dam, and an intact GA was necessary for EcoDam. Theoretical alignment of the region of best contacts between the protein and DNA showed that in the case of a palindromic interaction site, a zone covering the 5′-symmetric residues is located in the major groove versus a zone of contact covering the 3′-symmetric residues in the minor groove. Our data fit a simple rule of thumb that the most important contacts are aligned around the methylation target base: if the target base is in the 5′ half of the palindrome, the interaction between the enzyme and the DNA occurs mainly in the major groove; if it is in the 3′ half, the interaction occurs mainly in the minor groove.