Mutants of the N-3 R-Factor Conditionally Defective in hspII Modification and Deoxyribonucleic Acid-Cytosine Methylase Activity

The N-3 drug resistance (R) factor specifies a deoxyribonucleic acid (DNA)-cytosine methylase and a DNA restriction-modification (hspII) system. We have isolated three independent mutants that are conditionally defective in their ability to modify bacteriophage lambda and to methylate DNA-cytosine residues. The ratio of 5-methylcytosine to N(6)-methyladenine in bacterial DNA and in the DNA of phages lambda and fd was determined after labeling with [methyl-(3)H]methionine at various growth temperatures. Although the ability of the wild-type N-3 factor to modify phage lambda and to methylate DNA-cytosine residues was unaffected with increasing temperature, two of the mutants exhibited a parallel loss in modification and cytosine methylation ability. The ability of the third mutant to carry out these functions was dependent on the presence or absence of an amber suppressor mutation in the host genome. These results offer further support for the notion that hspII modification is mediated by a DNA-cytosine methylase. Evidence is also presented that the modification methylase is responsible for the in vivo methylation of phage fd DNA (which is not subject to hspII restriction in vivo).     

Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila.

The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA. In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3'. Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei. The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.     

Isolation of Deoxyribonucleic Acid Methylase Mutants of Escherichia coli K-12

Fourteen deoxyribonucleic acid (DNA) and 10 ribonucleic acid (RNA) methylation mutants were isolated from Escherichia coli K-12 by examining the ability of nucleic acids prepared from clones of unselected mutagenized cells to accept methyl groups from wild-type crude extract. Eleven of the DNA methylation mutants were deficient in 5-methylcytosine (5-MeC) and were designated Dcm. Three DNA methylation mutants were deficient in N(6)-methyladenine (N(6)-MeA) and were designated Dam. Extracts of the mutants were tested for DNA-cytosine:S-adenosylmethionine and DNA-adenine:S-adenosylmethionine methyltransferase activities. With one exception, all of the mutants had reduced or absent activity. A representative Dcm mutation was located at 36 to 37 min and a representative Dam mutation was located in the 60-to 66-min region on the genetic map. The Dcm mutants had no obvious associated phenotypic abnormality. The Dam mutants were defective in their ability to restrict lambda. None of the mutations had the effect of being lethal.     

Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation.

We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.     

Sequence specificity of DNA methylases from Bacillus amyloliquefaciens and Bacillus brevis

DNA methylation in Bacillus amyloliquefaciens strain H (Bam)² and Bacillus brevis (Bbv) has been examined by a variety of techniques. In vivo labelling studies revealed that Bam DNA contains no N⁶-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-³H]methionine ([³H]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 [³H]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-C1, C1-A and C1-T. This is consistent with a single, twofold symmetrical methylation sequence of 5′ … G-C1-(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-C1 and C1-T. We propose that this methylase is the BamI modification enzyme, and that the modified sequence is 5′ … G-G-A-T-C1-C … 3′.Bbv appears to contain two DNA-adenine methylases which produce the (partial) methylated sequences, 5′ … G-A1-T … 3′ and 5′ … A-A1-G … 3′, respectively; in the former case, all the G-A-T-C sites on Bbv DNA appear to be methylated.     

In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R. Eco RII).

We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R. Eco RII). The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E. coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd. mec+ RFI, isolated from the parental mec+ strain, is not cleaved. (ii) E. coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme.     

DNA-methylases from different strains of Bacillus subtilis and Bacillus natto

DNA-methyltransferase activity has been detected in some of Bacillus subtilis and Bacillus natto strains. Two strains of Bacillus subtilis exhibited DNA-cytosine methyltransferase activity, and the strains of Bacillus natto exhibited DNA-adenine methyltransferase activity. A possible effect of DNA-methyltransferase specificity on transformation efficiency is discussed.     

Isolation of a Mutant of Escherichia coli Defective in Cytosine-Specific Deoxyribonucleic Acid Methylase Activity and in Partial Protection of Bacteriophage λ Against Restriction by Cells Containing the N-3 Drug-Resistance Factor

A mutant (designated mec(-)) of Escherichia coli F(+) 100 endo I(-)su(+) r(K) (-)m(K) (+) has been isolated which is defective in cytosine-specific deoxyribonucleic acid (DNA) methylase activity. The DNA of this mutant, as well as the DNA of phages lambda and fd propagated in it, is virtually devoid of 5-methyl-cytosine (MeC); in contrast, the mutation has no significant effect on the level of N(6)-methyladenine in DNA. Phage lambda grown on the mec(-) mutant is more strongly restricted by N-3-containing cells than is lambda grown on the mec(+) parent. These results suggest that methylation of certain cytosine residues by the E. coli K-12 enzyme partially protects lambda DNA from either the N-3 restriction nuclease or against secondary degradation subsequent to N-3-specific degradation. Analysis of the MeC level in viral and cellular DNA obtained from mec(+), mec(+) (m(N3) (+)), and mec(-) (m(N3) (+)) strains has led to the conclusion that the R-factor controlled DNA-cytosine methylase may be capable of methylating a sequence(s) which is a substrate for the K-12 enzyme.     

Crosslinking of Dam methyltransferase with S-adenosyl-methionine

Highly purified DNA-adenine methyltransferase was irradiated in the presence of different concentrations of radiolabelled S-adenosyl-methionine (AdoMet) with a conventional Mineralight UV-lamp from several minutes up to 1 h while incubating in ice. Incorporation of radioactivity was monitored by electrophoresis of the crosslink between S-adenosyl-methionine and Dam methylase on SDS-polyacrylamide gels followed by fluorography. Crosslinking reached a maximum in presence of 10 microM S-adenosyl-methionine; it was inhibited in the presence of substances which competitively inhibit methylation of DNA by Dam methylase, like sinefungin or S-adenosyl-homocysteine, but not in the presence of non-inhibitors like ATP or S-isobutyl-adenosine. The crosslink obtained was resistant against a wide range of even drastic conditions commonly used in protein and peptide chemistry. Proteins, which do not bind S-adenosyl-methionine, as well as heat inactivated Dam methylase were not photolabelled. After limited proteolysis the radioactive label appeared only in certain of the peptides obtained. From Western blots carried out with polyclonal antibodies produced against a synthetic peptide corresponding in its sequence to amino acids 92-106 of the Dam methylase, the crosslinking of AdoMet could be tentatively mapped at a position after amino acid 106.     

Methylation of parental and progeny DNA strands in Physarum polycephalum

Although 5-methylcytosine comprises 4 to 8% of the cytosine residues in the major nuclear DNA of Physarum polycephalum (Evans &; Evans, 1970), only 1 % of the cytosine residues of progeny DNA become methylated during replication. Further methylation occurs during the same and subsequent mitotic cycles, so that 6 to 7 cycles after its synthesis, 5-methylcytosine comprises 5 to 7% of the DNA-cytosine residues of a single generation of DNA. The extent of methylation occurring during the S period has been measured by the determination of the specific activity of the precursor (S-adenosylmethionine) and the product (DNA-5-methylcytosine) and by comparison of the radioactivity in DNA-cytosine and DNA-5-methylcytosine after incorporation of [¹⁴C]deoxycytidine. Continuing methylation of parental DNA has been shown, by density shift experiments and by the conversion of prelabeled DNA-cytosine to DNA-5-methylcytosine. The DNA-5-methylcytosine once formed was found to be stable.     

Reduced methyl group acceptance of 1-beta-D-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

Reduced methyl group acceptance of 1-β-d-arabinofuranosylcytosine-containing DNA polymers

Previous studies have shown that 1-β-d-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.     

DNA methylation. Inhibition of de novo and maintenance methylation in vitro by RNA and synthetic polynucleotides

A partially purified HeLa cell DNA methylase will methylate a totally unmethylated DNA (de novo methylation) at about 3-4% the rate it will methylate a hemimethylated DNA template (maintenance methylation). Our evidence suggests that many, if not most, dCpdG sequences in a natural or synthetic DNA can be methylated by the enzyme. There is a powerful inhibitor of DNA methylase activity in crude extracts which has been identified as RNA. The inhibition of DNA methylase by RNA may indicate that this enzyme is regulated in vivo by the presence of RNA at specific chromosomal sites. The pattern of binding of RNA to DNA in the nucleosome structure and the DNA replication complex may determine specific sites of DNA methylation. An even more potent inhibition of DNA methylase activity is observed with poly(G), but not poly(C), poly(A), or poly(U). The only other synthetic polynucleotides studied which inhibit DNA methylation as well as poly(G) are the homopolymers poly(dC).poly(dG) and poly (dA).poly(dT). These results point out the unique importance of the guanine residue itself in the binding of the DNA methylase to dCpdG, the site of cytosine methylation. The surprising inhibition of the methylation reaction by poly(dA).poly(dT), which is itself not methylated by the enzyme, suggests the possible involvement of adjacent A and T residues in influencing the choice of sites of methylation by the enzyme.     

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.     

The Gene Cluster for Spectinomycin Biosynthesis and the Aminoglycoside-Resistance Function of spcM in Streptomyces spectabilis

The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine-specific DNA methylases and N-6 adenine-specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.     

In vitro methylation of DNA with Hpa II methylase.

The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine. The enzyme will methylate this sequence if both DNA strands are unmethylated or if only one strand is unmethylated. Conditions have been developed for producing fully methylated DNA from various sources. In vitro methylation of this site protects the DNA against digestion by the restriction enzyme Hpa II as well as the enzyme Sma I which recognizes the hexanucleotide sequence CCCGGG. These properties make this enzyme a valuable tool for analyzing methylation in eukaryotic DNA where the sequence CCGG is highly methylated. The activity of this methylase on such DNA indicates the degree of undermethylation of the CCGG sequence. Several examples show that this technique can be used to detect small changes in the methylation state of eukaryotic DNA.     

Decrease in DNA methylase activity during preimplantation development in the mouse.

During early mouse development, there are large-scale changes in DNA methylation. These changes may be due to the availability or stability of the enzyme, DNA methyltransferase (methylase), which is responsible for maintenance of DNA methylation. A microassay for methylase activity in preimplantation embryos shows that the level of maternally inherited enzyme is extremely high in the egg and that this activity is stable for the first three cleavage divisions. However, from the 8-cell to the blastocyst stage, there is a marked and absolute decrease in enzyme activity.     

Selenite: a good inhibitor of rat-liver DNA methylase

DNA methylase from rat liver was partially purified through a DEAE sephacel column and characterized in an in vitro assay with respect to time, protein, DNA and S-adenosylmethionine curves. The Km for S-adenosylmethionine was 2.5 microM. Sodium selenium inhibited the methylation of DNA in a dose dependent fashion when added to the assay. It was also demonstrated that selenite non-competitively inhibits rat-liver DNA methylase with a Ki of 6.7 microM. Dithiothreitol had no effect on selenite inhibition and increasing amounts of DNA did not alter the inhibition. However, increasing amounts of protein overcame the inhibition, suggesting that selenite is reacting with the DNA methylase protein. DNA methylase isolated from selenite treated animals had only 43% of the activity as enzyme from control rats. It appears that selenite is a good inhibitor of DNA methylase.     

Determination of DNA methylase activity in animal tissue extracts

An express method for measuring the level of in vitro DNA methylation in homogenates and nuclei from animal tissues as well as during initial steps of DNA methylase isolation and purification when methylase activity is low and hardly testable by other methods has been suggested. The method is based on the measuring the radioactivity incorporated in filter adsorbed DNA (acid-insoluble material) 3H-label from S-adenosile-L-methionine as a result of in vitro DNA methylation. The advantage of the method consists in the replacement of a long-duration repeated deproteinization procedure traditionally used by a relatively simple procedure (15 min incubation of the mixture at 80 degrees C with 10 volumes of the 8M urea, 5 mM EDTA, 5% n-butanol, 2% sodium dodecilsulfate, 1 M sodium chloride solution) and the absence of any loss of DNA. The method is fit for the fast serial assay of DNA methylase activity taking into consideration that about one third of the total acid-insoluble radioactivity is due to the radioactivity in 5-methylcytosine residues in DNA.