The effect of site-specific methylation on restriction-modification enzymes
Nucleic Acids ResearchPublished 1 January 1987Open access
Michael Nelson, Michael McClelland
Citations94
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EnergyBiochemistry, Genetics and Molecular Biology
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A procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA is presented and Restriction mapping and hybridization studies reveal the presence of closely linked beta-globin genes.
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It is shown that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as inDNA from Hemophilus influenzae and Escherichia coli and in the dam-3 mutant of E. coli.
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It is observed that plasmids containing certain cloned modification methylase genes of type II restriction-modification systems cannot be transformed into many laboratory strains of Escherichia coli K-12, and DNA containing 5-methylcytosine is biologically restricted by these strains, while DNA containing 6-methyladenine is not.
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222 Citations1977Michael B. Mann, Hamilton O. Smith
The methylases M. HaeIII and M.HpaII recognize the tetranucleotide sequences in DNA, and transfer a methyl group from S-adenosylmethionine to the 5-position of cytosine on each strand as indicated by the asterisks.
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Sea urchin (Echinus esculentus) DNA has been separated into high and low molecular weight fractions by digestion with the mCpG-sensitive restriction endonucleases Hpa II, Hha I and Ava I, and each of these gene families belongs to the unmethylated DNA compartment in all the tissues examined.
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It is proposed 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).
Journal of Biological ChemistryThe Deoxyribonucleic Acid Modification and Restriction Enzymes of Escherichia coli B
162 Citations1972James A. Lautenberger, Stuart Linn
Results imply that the termini of the restricted DNA have 3'-hydroxyl and 5'-phosphoryl groups, and that they have a duplex structure, however, restricted DNA cannot be phosphorylated by polynucleotide kinase, even after treatment of the DNA with alkaline phosphatase.
Nature New BiologyDNA Substrate Site for the EcoRII. Restriction Endonuclease and Modification Methylase
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The same nucleotide sequence in double stranded DNA restricted by the EcoRII restriction endonuclease is methylated by the ecoRII modification methylase, which generates DNA fragments with cohesive termini.
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126 Citations1977Richard Gelinas, Phyllis A. Myers +1 more
Two new sequence-specific endodeoxyribonucleases have been partially purified from Moraxella bovis and each cleave bacteriophage lambda DNA and adenovirus-2 DNA at more than 50 sites.
CellPatchwork structure of a bovine satellite DNA
121 Citations1979Michael Pech, Rolf E. Streeck +1 more
The nucleotide sequence of this satellite DNA in both cloned fragments and fragments from the total satellite DNA is determined, revealing both the overall extent of divergence and the frequency of base changes at individual positions of the 23 bp repeats are characteristically different in the various segments.
Nucleic Acids ResearchTwo restriction endonucleases from Bacillus globiggi
117 Citations1976Vincenzo Pirrotta
The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped and this enzyme makes staggered cuts producing sticky ends and produces 5' terminals which are not substrates for polynucleotide kinase.
Nucleic Acids ResearchThe effect of sequence specific DNA methylation on restriction endonuclease cleavage
117 Citations1981Michael McClelland
The known effects of sequence specific methylation on restriction endonucleases are compiled.
BiochemistryPhysical identity of the SV40 deoxyribonucleic acid sequence recognized by the Eco RI restriction endonuclease and modification methylase
117 Citations1974Achilles Dugaiczyk, Joe Hedgpeth +2 more
Proceedings of the National Academy of SciencesCloned restriction/modification system from Pseudomonas aeruginosa.
109 Citations1983T Gingeras, Joan E. Brooks
DNA fragments from Pseudomonas aeruginosa carrying the PaeR7 restriction/modification genes have been cloned in the plasmid vector pBR322 and propagated in Escherichia coli and evidence is presented that the PAER7 methylase modifies the adenine residue within the recognition sequence.
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106 Citations1977Gerald F. Vovis, Sanford A. Lacks
Two enzymes are complementary in that they recognize and cleave within the same DNA sequence, one if the DNA is methylated, the other if it is unmethylated, and these methylated hybrids were refractory to endonucleolytic cleavage by both endo R · DpnI and endo DpnII.
Nucleic Acids ResearchRestriction enzyme digestion of hemimethylated DNA
102 Citations1981Yosef Gruenbaum, Howard Cedar +1 more
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).
Molecular and General Genetics MGGRestriction and modification in B. subtilis
92 Citations1975Sierd Bron, Kenneth Murray +1 more
All Bacillus subtilis R-type strains showing the phenomena of restriction and modification contain an endonuclease that inactivates in vitro the biological activity of a variety of DNAs lacking R-specific modification, such as transfecting SPP1, SPO2 and ϕ105 DNA, and transforming B. subtILis 168-type DNA.
Journal of Molecular BiologyMethylation and cleavage sequences of the EcoP1 restriction-modification enzyme
89 Citations1979Brigitte Bächi, Jakob Reiser +1 more
The sites of both cleavage and methylation in simian virus 40 DNA are mapped and the enzyme EcoP1 methylation and cleavage site is determined.
Journal of Biological ChemistryCloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII.
89 Citations1983Roxanne Y. Walder, Carol J. Langtimm +2 more
None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked, and the relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.
Nucleic Acids ResearchThe effect of site specific methylation on restriction endonuclease digestion
88 Citations1985Michael McClelland, Michael Nelson
Methylasespecificities which partly overlap and block cleavage at endonuclease recognition sequences give restriction specificities which are both novel and rarer.
Proceedings of the National Academy of SciencesMethylation of the viral genome in an in vitro model of herpes simplex virus latency.
87 Citations1982Hagop Youssoufian, Scott M. Hammer +2 more
Results suggest a role for DNA methylation in the mechanism of HSV-1 latency in this in vitro model of latency in a lymphoid cell line developed recently.
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This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MSPI rather than a novel form of DNA methylation at this site in mammalian cells.
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The sequence methylated by NDA methylase II is consistent with the recognition site determined by Kelly and Smith (1970) for the H. influenzae restriction enzyme, endonuclease R.
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An in vitro method of altering the apparent cleavage specificities of restriction endonucleases was developed using DNA modification methylases and single-site cleavages were achieved for Nhe I in Adeno-2 DNA and for Acc I and Hinc II in pBR322 DNA by specifically methylating all but one recognition sequence.
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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 a sequence recognized by methylases specified by the dam or dcm gene.
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It is suggested that the K12 met+ cytosine methylase and the N-3 plasmid modification methylase are capable of recognizing the same nucleotide sequences, but that the in vivo methylation rate is lower in mec+ cells.
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67 Citations1985Nat Sternberg
In this review, most of the information presented will be derived from studies with Escherichia coli K-12 (E. coli) and its related bacteriophages, because more is known about methylation in these organisms than in any other.
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DNAs were prepared from twenty-six bacterial species and digested with a variety of restriction endonucleases to determine what modifications the DNAs carry.
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From the DNA sequencing of the plasmid pJD1 by a modification of the Maxam and Gilbert chemical cleavage procedure, the cytosine methylation specificities were demonstrated and five of these methylating enzymes and their respective specificities are M .
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The 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.
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In vivo labelling studies revealed that Bam DNA contains no N 6 -methyladenine, but contains 5-methylcytosine (MeCyt); approximately 0·7% of the cytosine residues are methylated.
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The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated and suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.
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The DNA-protein interactions involved in the recognition of a specific hexameric sequence, GAATTC, by the EcoRI modification methylase is examined by using derivatives of an oligodeoxyribonucleotide that contain a variety of base analogues, with results ranging from complete inactivity to enhanced reactivity.
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This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification.
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Experiments using a B. subtilis strain whose DNA is sensitive to HpaII and resistant to MspI degradation, indicated that methylation of the outer C of this sequence provides protection against the restriction enzyme MSPI.
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55 Citations1981Michael McClelland
M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT, and M.ThI protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.
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55 Citations1985Michael McClelland, Michael Nelson +1 more
The restriction modification methylase Mbo II has been purified using a sensitive oligonucleotide linker assay and the use of combinations of adenine methylases and Dpn I to generate highly selective DNA cleavages at a variety of sequences up to fourteen base pairs is discussed.
European Journal of BiochemistryRestriction and Modification in Bacillus subtilis. Localization of the Methylated Nucleotide in the BsuRI Recognition Sequence
55 Citations1978Ursula Günthert, K. Storm +1 more
Calf thymus DNA was methylated in vitro with cell extracts of Bacillus subtilis OG3R (r+m+) and S-adenosyl[Me-3H]methionine and revealed the following positions of the methylated nucleosides within the BsuRI recognition sequence.
Proceedings of the National Academy of SciencesSite-specific cleavage of DNA at 8- and 10-base-pair sequences.
51 Citations1984Michael McClelland, Louis G. Kessler +1 more
It is described how the above technique can be extended to generate Dpn I cleavage sites of up to 12 base pairs, and potential applications, including the development of cloning vectors and a rapid method for chromosome walking, are discussed.
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49 Citations1981Adinah Quint, Howard Cedar
The enzyme Hpa II methylase extracted and partially purified from Haemophilus parainfluenza catalyzes the methylation of the tetranucleotide sequence CCGG at the internal cytosine, indicating the degree of undermethylation of this sequence.
GeneSingle-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3A and TaqI
49 Citations1980Rolf E. Streeck
It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage, while a Sau3A site (GATC), however, is rendered resistant todouble-stranded cleavage by methylation of a single cytosines.
Proceedings of the National Academy of SciencesRecognition site of <i>Escherichia coli</i> B restriction enzyme on ϕXsB1 and simian virus 40 DNAs: An interrupted sequence
47 Citations1978James A. Lautenberger, Nancy C. Kan +4 more
Analysis of the sequence of varphiXam3cs70 showed that if no other residues are recognized, all seven of these bases are essential for recognition and the interval between the two groups of specified bases must be precisely eight.
Nucleic Acids ResearchMethylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs
46 Citations1986Richard Y.‐H. Wang, Xian‐Yang Zhang +4 more
Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence, which reveals a common 5'-m5CGRm5CG-3' element or closely related sequence.
Journal of BacteriologyRestriction and modification in Bacillus subtilis: sequence specificities of restriction/modification systems BsuM, BsuE, and BsuF
46 Citations1983Stefan Jentsch
The sequence specificities of three Bacillus subtilis restriction/modification systems were established, rendering such sites resistant to MspI degradation and leaving the majority of sites sensitive to HpaII degradation.
Journal of BacteriologyCytosine methylation of the sequence GATC in a mycoplasma
43 Citations1982Kevin Dybvig, David Swinton +2 more
It is shown that, in strain K2 and L2 virus grown on K2 cells, cytosine in the sequence GATC is methylated to 5-methylcytosine and, although strain K1 and L1 viruses grown onK2 contain N6-methyladenine in their DNA, adenine inthe sequence G ATC is not methylated.
The Journal of BiochemistryA DNA Methylase from Thermus thermophilus HB8
42 Citations1980Showbu Sato, Kayoko Nakazawa +1 more
A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium and probably recognizes the sequence, 5'-TCGA-3', in a double stranded DNA and probably methylates adenine in the above sequence.
Journal of Molecular EvolutionThe frequency and distribution of methylatable DNA sequences in leguminous plant protein coding genes
40 Citations1983Michael McClelland
An analysis of the distribution of di- and trinucleotides across functionally classified regions of genes showed CpG to be asymmetrically distributed, which may reflect a methylation-mediated regulatory role for this region in some legume genes.
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein SynthesisOn the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.
33 Citations1975B. F. Vanyushin, Anatoly P. Dobritsa
DNA in bacterial cells can be undermethylated because this enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences.
GeneDNA protection with the DNA methylase M · BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII
28 Citations1980Anatoly P. Dobritsa, Svetlana V. Dobritsa
The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.
Journal of Molecular BiologyDetection of methylated sequences in eukaryotic DNA with the restriction endonucleases SmaI and XmaI
24 Citations1981Hagop Youssoufian, Carel Mulder
The restriction endonuclease Xma I cleaves eukaryotic DNA whether or not its recognition sequence contains 5-methylcytosine in the CpG dinucleotide, in contrast to its isoschizomer, endo R Sma I, which does not cleave DNA if theCpG of its Recognition sequence is methylated.
FEBS LettersThree sequence‐specific endonucleases from <i>Escherichia coli</i> RFL47
21 Citations1983A. Janulaitis, M. Petrušyté +1 more
The characterization of the new restriction enzyme Eco47III recognizing a hexanucleotide palindromic sequence 5'AGC↓GCT and cleaving, as indicated by the arrow, is reported.
Nucleic Acids ResearchMethylation of either cytosine in the recognition sequence CGCG inhibits<i>Tha</i>I cleavage of DNA
19 Citations1984Jeannine S. Strobl, E. Brad Thompson
It is concluded that ThaI is a useful restriction enzyme for the analysis of mC at CGCG sequences in eukaryotic DNA.
FEBS Letters<i>Eae</i>I: a restriction endonuclease from <i>Enterobacter aerogenes</i>
18 Citations1983Philip R. Whitehead, Nigel L. Brown
EaeI may be useful in molecular cloning experiments, especially in conjunction with other enzymes which generate the same terminal extensions, including Enterobacter aerogenes, which generates DNA fragments with 5′‐tetranucleotide extensions.
Journal of VirologySelective protection of 5' ... GGCC ... 3' and 5' ... GCNGC ... 3' sequences by the hypermodified oxopyrimidine in Bacillus subtilis bacteriophage SP10 DNA
13 Citations1984C L Wiatr, H Witmer
In vitro results provide an explanation for the in vivo resistance of SP10 to restriction-modification by B. subtilis R. BsuRI and suggest ways in which the presence of the atypical base YThy in regions that flank the target might upset critical DNA-enzyme interactions necessary to locate and recognize the specific site of cleavage or methylation.
Biochemical JournalIsolation and characterization of the M.<i>Eae</i>I modification methylase
11 Citations1986David K. Jacobs, Nathan Brown
The modification enzyme corresponding to the restriction endonuclease EaeI was partially purified from Enterobacter aerogenes PW201 and protects overlapping 5'-C-C-G- G-3' sites against cleavage by both HpaII and MspI.
Nucleic Acids ResearchRestriction endonucleases from<i>Herpetosiphon giganteus</i>: an example of the evolution of DNA recognition specificity?
11 Citations1986Philip R. Whitehead, David K. Jacobs +1 more
The partial purification and characterisation of five Type II restriction endonucleases from two strains of Herpetosiphon giganteus are described and HgiJII was the first enzyme found that cleaves DNA at the family of related sequences 5'-G-R-G-C-Y/C-3'.
Annales de l Institut Pasteur MicrobiologieSusceptibility of Neisseria gonorrhoeae DNA to cleavage by restriction endonuclease KpnI
4 Citations1985M.F. Prère, Olivier Fayet
This work compared the susceptibility of Escherichia coli and Neisseria gonorrhoeae DNA to cleavage by KPNI and found that KpnI has a lower affinity for gonococcal DNA.
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