Recognition sequences of restriction endonucleases and methylases--a review

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.     

Avoidance of DNA methylation

Theocr ⁺ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5′-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzymeEcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonucleaseEcoRII.     

REBASE--restriction enzymes and methylases

REBASE contains comprehensive information about restriction enzymes, DNA methylases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal data and sequence data. Homing endonucleases are also included. Most recently, extensive information about the methylation sensitivity of restriction enzymes has been added and a new feature contains complete analyses of the putative restriction systems in the sequenced bacterial and archaeal genomes. The data is distributed via email, ftp (ftp.neb.com) and the Web (http://rebase. neb.com).     

Partial purification of the Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase: in vitro methylation completely protects bacteriophage lambda deoxyribonucleic acid against cleavage by R-EcoRII.

A procedure is described for the partial purification of the deoxyribonucleic acid (DNA)-cytosine methylases controlled by the RII plasmid and by the Escherichia coli mec+ gene. The two enzymes exhibit similar but distinct chromatographic behavior on diethylaminoethyl-cellulose and phosphocellulose. Preliminary studies on the two methylases indicate that they are indistinguishable with respect to their Km for S-adenosylmethionine and their pH (in tris (hydroxymethyl)aminomethane buffer) and NaCl concentration optima. In vitro methylation of various phage lambda DNA substrates by the mec'r RII enzyme modifies the DNA to a form that is completely resistant to double-stranded cleavage by the RII restriction endonuclease (R-EcoRII). These results are consistent with our earlier proposal that the mec8ethylase recognizes RII host specificity sites.     

Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI.

A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L. M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA). M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT. Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.     

Specificity of restriction endonucleases and methylases--a review

The properties and sources of all known restriction endonucleases and methylases are listed. The enzymes are cross-indexed (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the double-stranded DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (integrated into Table II), the structure of the generated fragment ends (Table III), and the sensitivity to different kinds of DNA methylation (Table V). In Table IV the conversion of two- and four-base 5'-protruding ends into new recognition sequences is compiled which is obtained by the fill-in reaction with Klenow fragment of the Escherichia coli DNA polymerase I or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments. Table VI classifies the restriction methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises restriction endonucleases which are known to be inhibited or activated by the modified nucleotides. The detailed sequences of those overlapping restriction sites are also included which become resistant to cleavage after the sequential action of corresponding restriction methylases and endonucleases [N11, M21]. By this approach large DNA fragments can be generated which is helpful in the construction of genomic libraries. The data given in both Tables IV and VI allow the design of novel sequence specificities. These procedures complement the creation of universal cleavage specificities applying class IIS enzymes and bivalent DNA adapter molecules [P17, S82].     

Avoidance of DNA methylation. A virus-encoded methylase inhibitor and evidence for counterselection of methylase recognition sites in viral genomes

The ocr+ gene of bacterial virus T7 codes for the first protein recognized to inhibit a specific group of DNA methylases. The recognition sequences of several other DNA methylases, not susceptible to Ocr inhibition, are significantly suppressed in the virus genome. The bacterial virus T3 encodes an Ado-Met hydrolase, destroying the methyl donor and causing T3 DNA to be totally unmethylated. These observations could stimulate analogous investigations into the regulation of DNA methylation patterns of eukaryotic viruses and cells. For instance, an underrepresentation of methylation sites (5'-CG) is also true for animal DNA viruses. Moreover, we were able to disclose some novel properties of DNA restriction-modification enzymes concerning the protection of DNA recognition sequences in which only one strand can be methylated (e.g., type III enzyme EcoP15) and the primary resistance of (unmethylated) DNA recognition sites towards type II restriction endonuclease EcoRII.     

A genetic and functional analysis of the unusually large variable region in the M·Alu I DNA-(cytosine C5 )-methyltransferase

The M·AluI DNA-(cytosine C5)-methyltransferase (5mC methylase) acts on the sequence 5′-AGCT-3′. The amino acid sequences of known 5mC methylases contain ten conserved motifs, with a variable region between Motifs VIII and IX that contains one or more “target-recognizing domains” (TRDs) responsible for DNA sequence specificity. Monospecific 5mC methylases are believed to have only one TRD, while multispecific 5mC methylases have as many as five. M·AluI has the second-largest variable region of all known 5mC methylases, and sequence analysis reveals five candidate TRDs. In testing whether M·AluI is in fact monospecific it was found that AGCT methylation represents only 80–90% of the methylating activity of this enzyme, while control experiments with the enzyme M·HhaI gave no unexplained activity. Because individual TRDs can be deleted from multispecific methylases without general loss of activity, a series of insertion and deletion mutants of the M·AluI variable region were prepared. All deletions that removed more than single amino acids from the variable region caused significant loss of activity; a sensitive in vivo assay for methylase activity based on McrBC restriction suggested that the central portion of the variable region is particularly important. In some cases, multispecific methylases can accommodate a TRD from another multispecific methylase, thereby acquiring an additional specificity. When TRDs were moved from a multispecific methylase into two different locations in the variable region of M·AluI, all hybrid enzymes had greatly reduced activity and no new specificities. M·AluI thus behaves in most respects as a monospecific methylase despite the remarkable size of its variable region.     

Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.     

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.     

Specificity of action of DNA methylases BcnI, CfrI and Cfr10I

The site specificity of three DNA methylases BcnI, CfrI and Cfr10I was determined to be 5'Cm4C(C/G)GG, 5'PyGGm5CCPu and 5'Pum5CCGGPy, respectively. Using the modification methylases under investigation with known restriction endonucleases, fourteen new DNA cleavage specificities can be created. Some aspects of the use of restriction endonucleases in DNA methylation analysis are discussed.     

Methylation of intact chromosomes by bacterial methylases in agarose plugs suitable for pulsed-field electrophoresis. Methylation of intact chromosomes in agarose by methylases

Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.     

Restriction analysis and quantitative estimation of methylated bases of filamentous and unicellular cyanobacterial DNAs.

The DNAs of strains of three cyanobacterial genera (Anabaena, Plectonema, and Synechococcus) were found to be partially or fully resistant to many restriction endonucleases. This could be due to the absence of specific sequences or to modifications, rendering given sequences resistant to cleavage. The latter explanation is substantiated by the content of N6-methyladenine and 5-methylcytosine in these genomes, which is high in comparison with that in other bacterial genomes. dcm- and dam-like methylases are present in the three strains (based on the restriction patterns obtained with the appropriate isoschizomeric enzymes). Their contribution to the overall content of methyladenine and methylcytosine in the genomes was calculated. Partial methylation of GATC sequences was observed in Anabaena DNA. In addition, the GATC methylation patterns might not have been random in the three cyanobacterial DNA preparations, as revealed by the appearance of discrete fragments (possibly of plasmid origin) withstanding cleavage by DpnI (which requires the presence of methyladenine in the GATC sequence).     

Four-stranded DNA structure and DNA base methylation in the mechanism of action of restriction endonucleases.

We examined the probability of short palindromec DNA sequences to occur as four-stranded structures held together in double-helical DNA by the additional hydrogen bonds postulated by McGavin (1971). The likeliness of the palindromes to be folded at their symmetry axes to allow the additional hydrogen bonding was considered using published physicochemical evidence and theoretical deductions. We deduced that both in vivo and in vitro the requirements may be met for duplex DNA folding which would approach palindrome complementary base bairs and thus allow the formation of the additional hydrogen bonds. However, we propose hydrogen bonding between guanine-cytosine base pairs to be different than that proposed by McGavin. Using CPK atom models we found that formation of the tertiary conformation already proposed by other authors and which we call the cage structure may be prevented or hindered by adenine, guanine or cytosine methylation. The available experimental data on recognition and cleavage site specificity of the Type 11 restriction endonucleases were confronted with the cage model as an alternative of the cruciform model and with the postulated effects of base methylation. The published data did not contradict the validity of the cage model and the role of base methylation in preventing the four-stranded palindrome structure. An applicability of the basic ideas of four-stranded DNA and base methylation effect to the mechanism of action of modification methylases and other restriction endonucleases was shortly discussed, but only tentative conclusions could be reached.     

Lack of DNA methylation in Schistosoma mansoni

Schistosoma mansoni genomic DNA from male and female adult worms was subjected to restriction by the isoschizomeric endonucleases HpaII and MspI, which display different sensitivities with respect to cytosine methylation. The digested DNA was hybridized with 13 S. mansoni probes. Southern blot analysis showed that there were no observable differences in the restriction patterns of the two isoschizomers and that the patterns were identical in male and female parasites. Adenine methylation was also ruled out since no differences were observed with DpnI, Sau3A1, or MboI restriction enzymes. The methylation-dependent restriction endonuclease McrBC, which cleaves DNA containing methylcytosine and will not cleave unmethylated DNA, did not digest S. mansoni genomic DNA. These results demonstrate that the genome of adult S. mansoni is not methylated.     

The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).     

Some peculiarities of phage DDVI-specific methylases]

The types of methylases are found in the cellular extract of Escherichia coli B, infected with phage DDVI. One of them is a cellular enzyme, which methylates adenine to form 6-methylaminopurine (6-MAP) and is repressed in the infected cell in vivo. The second type, which is not found in the non-infected cells, is specific for phage DDVI and induces the formation of 7-methylguanine (7-MG). Both enzymes recognize various sites, which accounts for the ratio 6-MAP/7-MG to vary in heterological DNAs between 2.07 in phage Sd DNA and 0.40 in phage DDII DNA. During in vitro incubation with homologous methylases phage DDVI DNA and especially phage T2 DNA are subjected to further methylation, which is probably indicative of their "undermethylation" in vivo. The DDVI-specific enzyme, similar to B-specific type, methylates DNA with a normal set of nitrogenous bases (phages Sd and DDII), as well as DNAs containing 5-oxymethylcytosine and glucose (phages T2 and DDVI). Both methylases under study use only native double-helical DNA as substrate and are strongly inhibited by S-adenosylhomocysteine. Phage DDVI Methylase is characterized by low stability.     

Isolation and Characterization of Chloroplast DNA from Chlorella ellipsoidea

A circular DNA molecule was isolated from chloroplasts of Chorella ellipsoidea. The DNA had a buoyant density of 1.695 grams per cubic centimeter (36% GC) and a contour length of 56 micrometers (175 kilobase pairs). The restriction endonuclease analysis gave the same size. Agarose gel electrophoretic patterns of chloroplast DNA digested by several restriction endonucleases were also presented. The digestion by the restriction enzymes, HpaII, MspI, SmaI, and XmaI revealed no appreciable methylation at CG sites in chloroplast DNA.