Restriction and modification in Bacillus subtilis: sequence specificities of restriction/modification systems BsuM, BsuE, and BsuF.

The sequence specificities of three Bacillus subtilis restriction/modification systems were established: (i) BsuM (CTCGAG), an isoschizomer to XhoI; (ii) BsuE (CGCG), an isoschizomer to FnuDII; and (iii) BsuF (CCGG), an isoschizomer to MspI, HpaII. The BsuM modification enzyme methylates the 3' cytosine of the recognition sequence. The BsuF modification enzyme methylates the 5' cytosine of the sequence, rendering such sites resistant to MspI degradation and leaving the majority of sites sensitive to HpaII degradation.     

Restriction enzyme BstZ17I is sensitive to cytosine methylation

The cleavage patterns of a subset of restriction enzymes are blocked or impaired when a methylated CpG is overlapped with either the 5' or 3' end of the canonical restriction site. BstZ17I restriction endonuclease is a blunt-end cutter, which recognises the hexanucleotide sequence GTA(downward arrow)TAC. In this report, I show that the BstZ17I restriction enzyme is sensitive to cytosine methylation. Using both in vitro-methylated episomal plasmids and lambdaDNA, I demonstrate that the BstZ17I restriction enzyme is sensitive to cytosine methylation that occurs 3' and/or 5' of the canonical recognition sequence.     

Presence of 5-methylcytosine in CC(A/T)GG sequences (Dcm methylation) in DNAs from different bacteria.

The presence of CC(A/T)GG sequences with methylated internal cytosine (Dcm methylation) was determined in DNA from different genera of eubacteria. This methylation was studied by using restriction enzymes EcoRII and BstNI, which cleave unmethylated or methylated CC(A/T)GG sequences. Dcm methylation was only detected in genera of the family Enterobacteriaceae closely related to Escherichia: Shigella, Citrobacter, Salmonella, and Klebsiella.     

Construction of a Neisseria gonorrhoeae MS11 derivative deficient in NgoMI restriction and modification.

We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.     

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.     

Construction and use of chimeric SPR/phi 3T DNA methyltransferases in the definition of sequence recognizing enzyme regions.

Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non-conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.     

Procaryotic and eucaryotic traits of DNA methylation in spiroplasmas (mycoplasmas).

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.     

Taq52 I, a novel and thermostable type II restriction endonuclease from the genus Thermus, recognising the pentanucleotide sequence GC(A or T)GC and cleaving DNA between the first and second bases of the recognition sequence: G decreased or reduced C(A or T)GC.

127 isolates of the genus Thermus, from neutral and alkaline hot water springs on four continents, have been screened for the presence of restriction endonuclease activity. An isolate (YS52) from Yellowstone National Park, USA, showed a high level of restriction endonuclease activity when a cell free extract was incubated with lambda phage DNA at 65 degrees C. A Type II restriction endonuclease (Taq52 I) has been partially purified from this isolate and the recognition and cleavage site determined. Taq52 I has a novel interrupted palindromic tetranucleotide recognition sequence GCWGC, where W can be either adenine (A) or thymine (T). It hydrolyses the phosphodiester bond in both strands of the substrate between the first and second bases of the recognition sequence: 5'G decreased or reduced CWGC3', producing three-base 5'-OH overhangs (sticky ends). The enzyme has a pH optimum of 7.0, requires 8 mM MgCl2 for maximum activity and is thermally stable, retaining full enzyme activity following incubation at 79 degrees C for 10 min. Taq52 I not only represents a new addition to the Type II restriction endonucleases, but also increases the small list of thermostable restriction endonucleases.     

Effect of CpG methylation on Msp I.

The restriction enzyme Msp I is inhibited by the presence of a methyl moiety at the external cytosine of the sequence CCGG, but is generally unaffected by methylation at the internal cytosine. At specific subsets of this sequence such as the hexanucleotide CCGGCC, however, methylation of the internal cytosine strongly inhibits Msp I digestion, leading to artifacts in the interpretation of DNA methylation analyses. Our results show, for instance, that the CCGG site at the 5' end of the human gamma globin gene, which was thought to be methylated at both the internal and external cytosines, is actually methylated only at the internal CpG residue.     

Determination of DNA sequences containing methylcytosine in Bacillus subtilis Marburg.

The methylcytosine-containing sequences in the DNA of Bacillus subtilis 168 Marburg (restriction-modification type BsuM) were determined by three different methods: (i) examination of in vivo-methylated DNA by restriction enzyme digestion and, whenever possible, analysis for methylcytosine at the 5' end; (ii) methylation in vitro of unmethylated DNA with B. subtilis DNA methyltransferase and determination of the methylated sites; and (iii) the methylatability of unmethylated DNA by B. subtilis methyltransferase after potential sites have been destroyed by digestion with restriction endonucleases. The results obtained by these methods, taken together, show that methylcytosine was present only within the sequence 5'-TCGA-3'. The presence of methylcytosine at the 5' end of the DNA fragments generated by restriction endonuclease AsuII digestion and the fact that in vivo-methylated DNA could not be digested by the enzyme XhoI showed that the recognition sequences of these two enzymes contained methylcytosine. As these two enzymes recognized a similar sequence containing a 5' pyrimidine (Py) and a 3' purine (Pu), 5'-PyTCGAPu-3', the possibility that methylcytosine is present in the complementary sequences 5'-TTCGAG-3' and 5'-CTCGAA-3' was postulated. This was verified by the methylation in vitro, with B. subtilis enzyme, of a 2.6-kilobase fragment of lambda DNA containing two such sites and devoid of AsuII or XhoI recognition sequences. By analyzing the methylatable sites, it was found that in one of the two PyTCGAPu sequences, cytosine was methylated in vitro in both DNA strands. It is concluded that the sequence 5'-PyTCGAPu-3' is methylated by the DNA methyltransferase (of cytosine) of B. subtilis Marburg.     

Cloning and characterization of the genes encoding the MspI restriction modification system.

The genes encoding the MspI restriction modification system, which recognizes the sequence 5' CCGG, have been cloned into pUC9. Selection was based on expression of the cloned methylase gene which renders plasmid DNA insensitive to MspI cleavage in vitro. Initially, an insert of 15 kb was obtained which, upon subcloning, yielded a 3 kb EcoRI to HindIII insert, carrying the genes for both the methylase and the restriction enzyme. This insert has been sequenced. Based upon the sequence, together with appropriate subclones, it is shown that the two genes are transcribed divergently with the methylase gene encoding a polypeptide of 418 amino acids, while the restriction enzyme is composed of 262 amino acids. Comparison of the sequence of the MspI methylase with other cytosine methylases shows a striking degree of similarity. Especially noteworthy is the high degree of similarity with the HhaI and EcoRII methylases.     

Cleavage at the twelve-base-pair sequence 5''-TCTAGATCTAGA-3'' using M.Xbal (TCTAGm6A) methylation and DpnI (Gm6A/TC) cleavage.

The DNA methylase M.Xbal was isolated from an E. coli recombinant clone. We deduce that the enzyme methylates at the sequence 5'-TCTAGm6A-3'. In combination with the methylation-dependent restriction endonuclease, DpnI (5'-Gm6A/TC-3'), DNA cleavage occurs at the sequence 5'-TCTAGA/TCTAGA-3'. This twelve-base-pair site should occur once every 16,000,000 base pairs in a random sequence of DNA. The exceptional rarity of the M.XbaI/DpnI sequence makes it an ideal candidate for transpositional integration of a unique cleavage site into bacterial genomes. Retrotransposition into mammalian genomes is also an attractive possibility.     

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.     

Plasmodium falciparum: evidence for a DNA methylation pattern

The methylation status of the adenine and cytosine residues in the genome of Plasmodium falciparum was studied using restriction enzymes exhibiting differential activity dependent on the methylation state of these residues in their recognition site. The gene coding for the enzyme dihydrofolate reductase-thymidylate synthase was studied for that purpose. No methylated adenine residues were observed in this gene in four strains tested. However, partial methylation of cytosine residues was observed in all strains. This methylation occurred at a specific site of the gene and was of the eukaryotic type, namely at a CpG sequence.     

DNA sequence selectivities in the covalent bonding of antibiotic saframycins Mx1, Mx3, A, and S deduced from MPE.Fe(II) footprinting and exonuclease III stop assays.

DNA sequence selectivities in the covalent binding of the antitumor antibiotic saframycins Mx1, Mx3, A, and S have been determined by complementary strand MPE.Fe(II) footprinting and exonuclease III stop assays on two different 545 and 135 base pair long HindIII/RsaI restriction fragments of pBR322 DNA. Saframycins Mx1, Mx3, A, and S recognize primarily 5'-GGG sequences. All four antibiotics also recognize 5'-GGPy sequences, however a cytosine is preferred over a thymine at the 3'-end of this recognition site in all cases. Saframycins Mx1, Mx3 and S, which possess the OH leaving group, also recognize the 5'-CCG sequence, in contrast to saframycin A, which contains the CN leaving group. In contrast, the OH-containing saframycins also recognize the 5'-CTA sequence. Saframycins Mx2, B and C, which lack the critical CN or OH leaving group, do not show any footprints on the restriction fragments examined in this study. The measured binding site size for all four antibiotics is three base pairs. The exonuclease III stop assay independently confirmed the formation of a covalent bond and the strong preference of the antibiotics for 5'-GGG and 5'-GCC sequences. The latter enzyme assay also suggests that the 5'-terminal or central G of the triad binding site is the base to which reversible covalent attachment of the antibiotic takes place.     

Gene cloning, comparative analysis of the protein structures from Fsp4HI restriction-modification system and biochemical characterization of the recombinant DNA methyltransferase

Genes coding for the restriction-modification system Fsp4HI, recognizing the sequence 5'-GCNGC-3' have been cloned in Escherichia coli ER2267 cells and its primary structure has been determined. This RM system consists of two genes: the DNA-methyltransferase gene which is followed by the restriction endonuclease gene in the same direction. The analysis of amino acid sequences of the proteins showed that M.Fsp4HI belongs to C5 DNA-methyltransferases, and the restriction enzyme shares more or less significant homology to just a few restriction endonucleases with related recognition sequences. M.Fsp4HI enzyme was purified by means of column chromatography. According to the results of biochemical study it was considered that M.Fsp4HI has its optimal activity at 30 degree C and pH 7.5. M.Fsp4HI modifies the first cytosine residue in the sequence 5'-GCNGC-3'.     

Methylation of mosquito DNA.

Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 mol% (A + T) and has about 0.03 mol% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.     

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.     

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.     

A cautionary note on the use of certain restriction endonucleases with methylated substrates

Methylation of adenine and cytosine residues in DNA isolated from common strains of Escherichia coli K-12 can render that DNA resistant to cleavage by certain restriction endonucleases at those sites at which the recognition sequence for such an endonuclease overlaps (but does not include) a sequence recognized by methylases specified by the dam or dcm gene.