Stanley Hattman

Sequence specificity of the P1 modification methylase (M·Eco P1) and the DNA methylase (M·Eco dam) controlled by the Escherichia coli dam gene

Abstract Labeled oligonucleotides have been fractionated from pancreatic DNase digests of DNA that had been methylated in vitro with the P1 modification enzyme (M· Eco P1) or with the DNA-adenine methylase (M· Eco dam) controlled by the Escherichia coli dam gene. The sequences of methylated oligonucleotides were established for M· Eco dam modification of calf thymus DNA. The results show that M· Eco dam inethylates adenine residues contained in the twofold symmetrical sequence, 5′ … G-A-T-C … 3′. The sequence for the site methylated by M· Eco P1 has also been deduced; we propose that M· Eco P1 modification produces the following methylated pentameric sequence: 5′ … A-G-A ∗ -C-Py … 3′ (where A ∗ = N 6 methyladenine and Py is C or T).

Transition from Nonspecific to Specific DNA Interactions along the Substrate-Recognition Pathway of Dam Methyltransferase

DNA methyltransferases methylate target bases within specific nucleotide sequences. Three structures are described for bacteriophage T4 DNA-adenine methyltransferase (T4Dam) in ternary complexes with partially and fully specific DNA and a methyl-donor analog. We also report the effects of substitutions in the related Escherichia coli DNA methyltransferase (EcoDam), altering residues corresponding to those involved in specific interaction with the canonical GATC target sequence in T4Dam. We have identified two types of protein-DNA interactions: discriminatory contacts, which stabilize the transition state and accelerate methylation of the cognate site, and antidiscriminatory contacts, which do not significantly affect methylation of the cognate site but disfavor activity at noncognate sites. These structures illustrate the transition in enzyme-DNA interaction from nonspecific to specific interaction, suggesting that there is a temporal order for formation of specific contacts.

DNA-[adenine] methylation in lower eukaryotes.

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase ((MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.

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 β-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.

DNA methylation of T-even bacteriophages and of their nonglucosylated mutants: Its role in P1-directed restriction

Abstract The 6-methylaminopurine (MAP) content of bacteriophages T2, T4, T6 and their nonglucosylated gt mutants has been analyzed. Phage T2 contains about 25% more MAP than T4; the nonglucosylated forms of T2 and T4 contain 30–100% more MAP than their respective wild-type parents; the level of MAP is affected by the growth temperature and by the level of glucosylation. T6 and its nonglucosylated mutants are devoid of MAP. Support for the hypothesis that methylation has a role in P1-directed restriction of gt mutants of T-even phages ( Revel and Georgopoulos, 1969 ) is presented: (1) mutants of T2gt and T4gt insensitive to P1-restriction, designated uP1 ( Revel and Georgopoulos, 1969 ) contain hypermethylated DNA; (2) cells simultaneously infected with P1-sensitive T2gt or T6gt (designated rP1) and ultraviolet-irradiated T2gt uP1 yield progeny rP1 phage which contain hypermethylated DNA and are partially resistant to P1 restriction. It is proposed that the uP1 mutations occur in the structural gene for phage DNA methylase. Methylation does not appear to be involved in the restriction of T2gt and T4gt by certain bacterial hosts that do not restrict T6gt (these bacteria are designated r6−r2,4+ because of the findings that (1) a T4gt mutant lacking MAP is still restricted in r6−r2,4+; (2) phenotypically methylated T6gt is accepted in r6−r2,4+

Characterization of viruses infecting a eukaryotic Chlorella-like green alga☆

Nineteen plaque-forming viruses of the unicellular, eukaryotic Chlorella-like green alga, strain NC64A, were isolated from various geographic regions in the United States and characterized. Like the previously described virus, PBCV-1, all of the new viruses were large polyhedrons, sensitive to chloroform, and contained large dsDNA genomes of ca. 300 kbp. All of the viral DNAs contained 5-methyldeoxycytidine which varied from 0.1 to 47% of the deoxycytidine. In addition, 10 of the viral DNAs contained N6-methyldeoxyadenosine which varied from 8.1 to 37% of the deoxyadenosine. These viruses, along with 11 previously described viruses which replicate in the same Chlorella host, were grouped into 11 classes based on at least one of the following properties: plaque size, reaction with PBCV-1 antiserum, or the nature and abundance of methylated bases in their genomic DNA.

Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase

Phages T2 and T4 induce synthesis of a DNA-adenine methylase which is coded for by a phage gene, dam+. These enzymes methylate adenine residues in specific sequences which include G-A-T-C, the methylation site of the host Escherichia coli dam+ methylase. Methylation of G-A-T-C to G-m6A-T-C protects the site against cleavage by the MboI restriction nuclease. We have taken advantage of this property to enrich and screen for transformants which contain a cloned, functional T4 dam+ gene. These recombinant molecules consist of a 1.85-kb HindIII fragment inserted into the plasmid pBR322; both orientations of the fragment express the methylase gene, suggesting that transcription is from a T4 promoter. We have tested the 1.85-kb insert for sensitivity to a variety of restriction nucleases and have found single sites for EcoRI, BalI, XbaI, and at least two sites for BstNI (EcoRII). The relative positions of these restriction sites have also been determined. Physical mapping was carried out by Southern blot hybridization with 32P-labeled (nick-translated clone) probe. These experiments showed that the insert corresponds to a HindIII fragment located on the physical map of T4 between positions 16.2 and 18.1 kb from the T4rIIA-rIIB junction. E. coli dam- possesses several phenotypic differences from the wild-type dam+ parent, including an increased sensitivity to 2-aminopurine (2-AP). We found that T4 dam+ clones could relieve dam- cells of their increased sensitivity to 2-AP.

Regulation and expression of the bacteriophage Mu mom gene: mapping of the transactivation (Dad) function to the C region

Expression of the bacteriophage Mu mom gene is under tight regulatory control. One of the factors required for mom gene expression is the trans-acting function (designated Dad) provided by another Mu gene. To facilitate studies on the signals mediating mom regulation, we have constructed a mom-lacZ fusion plasmid which synthesizes beta-galactosidase only when the Mu Dad transactivating function is provided. lambda pMu phages carrying different segments of the Mu genome have been assayed for their ability to transactivate beta-galactosidase expression by the fusion plasmid. The results of these analyses indicated that the Dad transactivation function is encoded between the leftmost EcoRI site and the lys gene of Mu; this region includes the C gene, which is required for expression of all Mu late genes. Cloning of an approx. 800-bp fragment containing the C gene produced a plasmid which could complement MuC- phages for growth and could transactivate the mom-lacZ fusion plasmid to produce beta-galactosidase. These results suggest that the C gene product mediates the Dad transactivation function.

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding

Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet [10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.

Sequence specificity of DNA methylases from Bacillus amyloliquefaciens and Bacillus brevis

Abstract DNA methylation in Bacillus amyloliquefaciens strain H ( Bam ) † and Bacillus brevis ( Bbv ) has been examined by a variety of techniques. In vivo labelling studies revealed that Bam DNA contains no N 6 -methyladenine (MeAde), but contains 5-methylcytosine (MeCyt); approximately 0·7% of the cytosine residues are methylated. DNA methylase activity was partially purified from both Bam and Bbv ; the Bam enzyme preparation transferred methyl groups from S -adenosyl- l -[ methyl - 3 H]methionine ([ 3 H]AdoMet) to specific DNA cytosine residues only; in agreement with Vanyushin & Dobritsa (1975), the Bbv enzyme preparation methylated both DNA adenine and cytosine residues. The (partial) sequence specificity of the methylases was determined by analyzing [ 3 H]methyl-labelled dinucleotides obtained from enzymatic digests of DNA methylated in vitro . Bam and Bbv each contain a DNA-cytosine methylase with overlapping sequence specificity; e.g. both enzymes produce G-C∗, C∗-A and C∗-T. This is consistent with a single, twofold symmetrical methylation sequence of 5′ … G-C∗-(A or T)-G-C … 3′; this was observed by Vanyushin & Dobritsa (1975) for a different Bbv strain. Bam contains a second DNA-cytosine methylase (not present in Bbv ), which produces T-C∗ and C∗-T. We propose that this methylase is the Bam I modification enzyme, and that the modified sequence is 5′ … G-G-A-T-C∗-C … 3′. Bbv appears to contain two DNA-adenine methylases which produce the (partial) methylated sequences, 5′ … G-A∗-T … 3′ and 5′ … A-A∗-G … 3′, respectively; in the former case, all the G-A-T-C sites on Bbv DNA appear to be methylated.