DNA methylase activities in a Neisseria gonorrhoeae extract

Abstract Resistance of Neisseria gonorrhoeae DNA to cleavage by various restriction endonucleases suggests the existence of modification enzymes which protect the corresponding recognition sequences. We indeed found methylase activities in N. gonorrhoeae extracts. These activities lead to the methylation of adenine and cytosine residues in bacteriophage λ DNA and DNA from an Escherichia coli Dam ⁻ strain. They also result in partial protection of λ DNA to cleavage by the restriction endonucleases Hae II, Hae III, Bam HI and, Sac II.     

Restriction enzyme digestion of hemimethylated DNA.

Hemimethylated duplex DNA of the bacteriophage phi X 174 was synthesized using primed repair synthesis is in vitro with E. coli DNA polymerase I followed by ligation to produce the covalently closed circular duplex (RFI). Single-stranded phi X DNA was used as a template, a synthetic oligonucleotide as primer and 5-methyldeoxycytidine-5'-triphosphate (5mdCTP) was used in place of dCTP. The hemimethylated product was used as substrate for cleavage by various restriction enzymes. Out of the 17 enzymes tested, only 5 (BstN I, Taq I, Hinc II, Hinf I and Hpa I) cleaved the hemimethylated DNA. Two enzymes (Msp I and Hae III) were able to produce nicks on the unmethylated strand of the cleavage site. Msp I, which is known to cleave at CCGG when the internal cytosine residue is methylated, does not cleave when both cytosines are methylated. Another enzyme, Apy I, cleaves at the sequence CCTAGG when the internal cytosine is methylated, but is inactive on hemimethylated DNA in which both cytosines are methylated. Hemimethylated molecules should be useful for studying DNA methylation both in vivo and in vitro.     

Sites of methylation of purified transfer ribonucleic acid preparations by enzymes from normal tissues and from tumours induced by dimethylnitrosamine and 1,2-dimethylhydrazine

1. The sites within the tRNA sequence of nucleosides methylated by the action of enzymes from mouse colon, rat kidney and tumours of these tissues acting on tRNA(Asp) from yeast and on tRNA(Glu) (2), tRNA(fMet) and tRNA(Val) (1) from Escherichia coli were determined. 2. The same sites in a particular tRNA were methylated by all of these extracts. Thus tRNA(Glu) (2) was methylated at the cytidine residue at position 48 and the adenosine residue at position 58 from the 5'-end of the molecule; tRNA(Asp) was methylated at the guanosine residue at position 26 from the 5'-end of the molecule; tRNA(fMet) was methylated at the guanosine residues 9 and 27, the cytidine residue 49 and the adenosine residue 59 from the 5'-end; tRNA(Val) (1) was methylated at the guanosine residue 10, the cytidine residue 48 and the adenosine residue 58 from the 5'-end. 3. All of these sites within the clover leaf structure of the tRNA sequence are occupied by a methylated nucleoside in some tRNA species of known sequence. It is concluded that methylation of tRNA from micro-organisms by enzymes from mammalian tissues in vitro probably does accurately represent the specificity of these enzymes in vivo. However, there was no evidence that the tumour extracts, which had considerably greater tRNA methylase activity than the normal tissues, had methylases with altered specificity capable of methylating sites not methylated in the normal tissues.     

The effects of DNA methylation by Hha I methylase on the cleavage reactions by Hae II, Aha II and Ban I endonucleases

The DNA methylated by Hha I methylase was resistant against cleavage of Hae II or Aha II endonuclease indicating that the methyl group of the C5 position of the inmost cytosine nucleotide interferes with the interaction between the enzyme and the hexameric recognition sequence. Considering that Hae II or Aha II methylase has not been isolated yet, the result explained above is a useful information for protecting a double stranded DNA from being cleaved by Hae II or Aha II endonuclease. In contrast to Hae II or Aha II endonuclease, Ban I endonuclease which also has Hha I sequence as its tetrameric core was able to cleave the same DNA normally. This result suggests that the C5 position of the inmost pyrimidine nucleotide is not an important contact point between Ban I endonuclease and its hexameric recognition sequence.     

Arthrobacter viscosus DNA is modified at GATC sequence

Arthrobacter viscosus DNA was resistance to digestion by restriction enzymes that are sensitive to methylation of the cytosine residue (but not of adenine) within the GATC recognition sequence. Restriction enzymes sensitive to methylation of cytosine in other recognition sequences were not affected. A. viscosus DNA thus appeared to contain methylated cytosine specifically at the GATC sequence.     

Extensive methylation of chloroplast DNA by a nuclear gene mutation does not affect chloroplast gene transmission in chlamydomonas

Based on analysis by high pressure liquid chromatography, greater than 35% of the cytosine residues in chloroplast DNA of vegetative cells were found to be methylated constitutively in the nuclear gene mutation (me-1) of Chlamydomonas reinhardtii, which has an otherwise wild-type phenotype. Digestion of chloroplast DNA from vegetative cells and gametes of this mutant with restriction endonucleases Hpa II and Msp I reveals that in the 5′CCGG3′ sequence, CpG is methylated extensively, whereas CpC is only methylated occasionally. Hae III (5′GGCC3′) digestion of the mutant chloroplast DNA also shows extensive methylation of the GpC sequence. In contrast to the results of Sager and colleagues, which show a correlation between methylation of chloroplast DNA and transmission of chloroplast genes in crosses, our results with crosses of the me-1 mutant suggest that extensive chloroplast DNA methylation may be insufficient to account for the pattern of inheritance of chloroplast genes in Chlamydomonas.     

Methylation of either cytosine in the recognition sequence CGCG inhibits ThaI cleavage of DNA.

ThaI (CGCG) sites which overlap HhaI (GCGC) sites in phi X174 and pBR322 DNA were methylated in vitro with HhaI methylase and S-adenosylmethionine to yield CGmCG, mCGCG or mCGmCG (5-methylcytosine, mC). Methylation of either cytosine in the ThaI recognition sequence rendered the DNA resistant to ThaI cleavage. Rat pituitary cell genomic DNA was digested with ThaI or 2 other known methylation-sensitive enzymes, AvaI or XhoI. After electrophoresis and ethidium bromide straining of the DNA, all 3 enzymes showed the infrequent DNA cleavage characteristic of methylation-sensitive enzymes. Comparison of pituitary growth hormone (GH) genes bearing strain-specific degrees of methylation showed the less methylated gene to be more frequently cut by either AvaI or ThaI. ThaI resistant sites in GH genes were cleaved by ThaI after exposing cells to 5-azacytidine, an inhibitor of DNA methylation. We conclude that ThaI is a useful restriction enzyme for the analysis of mC at CGCG sequences in eukaryotic DNA.     

Factors involved in the transformation of previously non-transformable Clostridium perfringens type B

Abstract The pre-shock incubation of cells plus DNA and the methylation state of plasmid DNA were found to play a role in the electroporation-based transformation of Clostridium perfringens 3626B. Following pre-shock incubation, the highest number of C. perfringens 3626B transformants was obtained when plasmid pGK201 was both dam⁺ dcm⁺ modified, while no transformants were obtained when pGK201 was not methylated or only dcm methylated. This is consistent with the observation that plasmid pGK201 was protected against digestion by C. perfringens 3626B cell-associated nucleases for up to 3 min when methylated by both methylases. C. perfringens 3626B was successfully transformed only within a narrow cell recovery rate window. The erm AM gene associated with pGK201 and pAK102 was found to integrate into the chromosome of C. perfringens strains 13A and 3626B.     

Sse8387I, a useful eight base cutter for mammalian genome analysis (influence of methylation on the activity of Sse8387I).

To develop restriction enzymes that are useful for genome analysis, we previously performed screening and isolated Sse8387I from Streptomyces sp. strain 8387. Sse8387I is a restriction enzyme that recognizes 5'-CCTGCA/GG-3' and cleaves DNA at the site shown by the diagonal (Nucleic Acid Res., 18, 5637-5640). The present study evaluated the effects of methylation that is important when Sse8387I is used for genome analysis. Sse8387I lost cleavage activity after methylation of adenine or methylation of cytosine at any site in the recognition sequence. However, the recognition sequence of Sse8387I contains no CG sequence, which is the mammalian methylation sequence. In addition, we evaluated the effects of methylation of CG at sites other than the recognition sequence. The cleavage activity of Sse8387I was maintained even when CG sequences were present immediately before or after, or near the recognition sequence, and cytosine was methylated. These results suggest that CG methylation does not affect the cleavage activity of Sse8387I. Therefore, Sse8387I seems to be very useful for mammalian genome analysis.     

Restriction endonuclease BsoFI is sensitive to the 5'-methylation of deoxycytidines in its recognition sequence.

The isoschizomeric restriction endonucleases Fnu4HI and BsoFI cleave DNA at 5'-GCdecreasesNGC-3' sequences. Fnu4HI has been shown to be inhibited by 5'-CG-3'methylation in the sequences 5'-GmCGGC-3' or 5'-GCGGmCG-3'. We have now investigated the methylation sensitivity of BsoFI by testing its activity on plasmid DNA 5'-CG-3' methylated with the M.SssI DNA methyltransferase or on synthetic (CGG)n repetitive oligodeoxyribonucleotides which have been partly or completely C methylated. The data demonstrate that BsoFI cannot cleave at its recognition sequence when it is completely 5'-CG-3' methylated. These enzymes have proven to be useful in analyses of the methylation status in (CGG)n repeats of the human genome.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

Abstract The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EII^Man) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EII^Man showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EII^Glc). Similar homology was shown between the C-terminal sequence of EII^Man and the E. coli glucose-specific enzyme III (EIII^Glc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EII^Man contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIII^Glc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

13C relaxation studies of the DNA target sequence for hhai methyltransferase reveal unique motional properties

The goal of this work was to examine if sequence-dependent conformational flexibility in DNA plays a role in base extrusion, a common conformational change induced by many DNA-modifying enzymes. We studied the dynamics of the double-stranded DNA target of the HhaI methyltransferase by recording an extensive set of (13)C NMR relaxation parameters. We observe that the cytidine furanose rings experience fast (picosecond to nanosecond) motions that are not present in other nucleotides; the methylation site experiences particularly high mobility. We also observe that the bases of guanosine and cytidine residues within the HhaI recognition sequence GCGC experience motions on a much slower (1-100 micros) time scale. We compare these observations with previous solution and solid-state NMR studies of the EcoRI nuclease target sequence, and solid-state NMR studies of a similar HhaI target construct. While an increased mobility of cytidine furanose rings compared to those of other nucleotides is observed for both sequences, the slower motions are only observed in the HhaI target DNA. We propose that this inherent flexibility lowers the energetic barriers that must occur when the DNA binds to the HhaI methyltransferase and for extrusion of the cytidine prior to its methylation.     

PROTEIN METHYLATION BY CEREBRAL TISSUE

Abstract— Transfer of the methyl group of S -adenosyl [Me-¹⁴C]methionine into cerebral proteins, an encephalitogenic protein and histones was studied using extracts of bovine and rat brains. The brain extract contains multiple substrate proteins and their lysine and arginine residues were methylated to form N^e-mono-, -di- and -trimethyl-lysine and N ^G-mono-, N ^G, N ^G- and N^G,N^G-dimethylarginine residues respectively, at different rates. The enzyme which catalyses the methylation of arginine residues was differentiated by ammonium sulphate fractionation from that methylating lysine residues. Methylation of arginine and lysine residues of proteins was stepwise in general, from mono- to dimethyl-arginine and from mono- to di- and trimethyl-lysines. Two different enzymes which methylate histone and the encephalitogenic basic protein respectively were obtained from the cytoplasmic fraction of rat brain and their enzymic properties were examined.     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

Additional Restriction Endonuclease Cleavage Sites on the Bacteriophage P22 Genome

We present complete restriction endonuclease cleavage site maps of the bacteriophage P22 chromosome for 16 enzymes with six base recognition sequences, thereby positioning 116 new sites on the chromosome. Twenty-four such restriction maps for P22 DNA, containing 162 sites, have now been completed, and three enzymes were found that did not cut P22 DNA. Our results are consistent with the ideas that ClaI does not cleave the methylated recognition sequence ATCGA(me)T or A(me)TCGAT and StuI does not cleave the methylated recognition sequence AGGCC(me)T.     

Tissue specific methylation of human Y chromosomal DNA sequences

This report describes two moderately repetitive human Y chromosomal DNA sequences isolated from a flow sorted Y chromosonal library. These sequences are present in XY male and XY female DNAs but absent in XX male and XX female DNAs. Genomic Southern blot analysis against DNAs isolated from different tissues showed tissue specific DNA methylation patterns. In contrast to the 2.1 kb Hae III repeats which are hypomethylated in sperm DNA, the moderately repetitive sequences used in this study are highly methylated in sperm, less methylated in blood and brain and least methylated in placental DNA.     

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI.

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.     

Restriction endonuclease isoschizomers ItaI, BsoFI and Fsp4HI are characterised by differences in their sensitivities to CpG methylation.

BsoFI , ItaI and Fsp4HI are isoshizomers of Fnu4HI (5'-GC NGC-3'). Both Fnu4HI and BsoFI have previously been shown to be inhibited by cytosine-specific methylation within the recognition sequence. Fnu4HI is inhibited if either the internal cytosine at position 2 or the external cytosine at position 5 of the restriction sequence is methylated, but the precise nature of the methylation sensitivity of BsoFI is unclear from the literature. The methylation sensitivities of ItaI and Fsp4HI have not previously been reported. By methylating the plasmid pUC18 with M.SssI (a DNA cytosine-5'-methyltransferase with a specificity for CpG), we have determined that ItaI is sensitive only to methylation of internal CpG sites within the restriction sequence. The methylation sensitivity of Fsp4HI is identical to that of Fnu4HI, being inhibited by methylation of either internal CpG sites or overlapping CpG sites. BsoFI , like the other isoschizomers tested, is sensitive to a combination of internal and overlapping CpG methylation. BsoFI is also sensitive to overlapping CpG methylation (in the absence of internal CpG methylation) if CpG overlap with both sides of the recognition sequence. Sites containing one overlapping CpG (in the absence of internal CpG) are cut when methylated but show marked individual variation in their rates of cleavage. Considerable variation in the rate of cleavage by BsoFI is also observed at sites containing only internal methylated CpG. Some sites are cut slowly, whilst others fail to cut even after prolonged incubation with excess of enzyme.     

The effect of methylation outside the recognition sequence of restriction endonuclease PvuII on its cleavage efficiency.

This study is to extend our earlier observation that Dam and Dcm methylation outside the PvuII recognition sequence inhibited PvuII cleavage in one of the three PvuII sites of pGEM4Z-ras DNA. In this paper, a new recombinant plasmid DNA, pGEM4-SV40ori-anti-ras, was constructed which has only two PvuII sites, I and II. The Dam and Dcm-methylated and unmethylated DNAs were produced in Escherichia coli and linearized by ScaI. The DNA molecules were digested with different amounts of PvuII. The results show that by comparing the DNA fragment number and intensity of the partial and final products in agarose gel, PvuII site I on the methylated DNA molecule was digested four- to eight-fold more slowly than site II. In the unmethylated plasmid DNA, the two PvuII sites were cleaved at about the same rate. The difference was caused only by methylation of Dam and Dcm sites outside the PvuII recognition sequence. A methylated Dam site immediately adjacent to the less efficiently cut PvuII site I may be responsible for the inhibitory effect. We suggest that a new parameter, involving methylation of sites outside the recognition sequence, be considered in kinetic experiments on cleavage.