Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1977 Mar;129(3):1330–1334. doi: 10.1128/jb.129.3.1330-1334.1977

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.

S Hattman
PMCID: PMC235106  PMID: 14921

Abstract

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.

Full text

PDF
1331

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bouché J. P., Dubert J. M. DNA methylases of Escherichia coli K12. Evidence for changes in their state of association following purification. Eur J Biochem. 1972 May;27(1):53–59. doi: 10.1111/j.1432-1033.1972.tb01810.x. [DOI] [PubMed] [Google Scholar]
  2. Boyer H. W., Chow L. T., Dugaiczyk A., Hedgpeth J., Goodman H. M. DNA substrate site for the EcoRII restriction endonuclease and modification methylase. Nat New Biol. 1973 Jul 11;244(132):40–43. doi: 10.1038/newbio244040a0. [DOI] [PubMed] [Google Scholar]
  3. Fujimoto D., Srinivasan P. R., Borek E. On the nature of the deoxyribonucleic acid methylases. Biological evidence for the multiple nature of the enzymes. Biochemistry. 1965 Dec;4(12):2849–2855. doi: 10.1021/bi00888a041. [DOI] [PubMed] [Google Scholar]
  4. GOLD M., HURWITZ J. THE ENZYMATIC METHYLATION OF RIBONUCLEIC ACID AND DEOXYRIBONUCLEIC ACID. V. PURIFICATION AND PROPERTIES OF THE DEOXYRIBONUCLEIC ACID-METHYLATING ACTIVITY OF ESCHERICHIA COLI. J Biol Chem. 1964 Nov;239:3858–3865. [PubMed] [Google Scholar]
  5. Hattman S., Gold E., Plotnik A. Methylation of cytosine residues in DNA controlled by a drug resistance factor (host-induced modification-R factors-N 6 -methyladenine-5-methylcytosine). Proc Natl Acad Sci U S A. 1972 Jan;69(1):187–190. doi: 10.1073/pnas.69.1.187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hattman S. Plasmid-controlled variation in the content of methylated bases in bacteriophage lambda deoxyribonucleic acid. J Virol. 1972 Sep;10(3):356–361. doi: 10.1128/jvi.10.3.356-361.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hattman S., Schlagman S., Cousens L. Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in partial protection of bacteriophage lambda against restriction by cells containing the N-3 drug-resistance factor. J Bacteriol. 1973 Sep;115(3):1103–1107. doi: 10.1128/jb.115.3.1103-1107.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hughes S. G. The sensitivity of bacteriophage lambda DNA to restriction endonuclease RII. J Mol Biol. 1975 Nov 5;98(3):645–647. doi: 10.1016/s0022-2836(75)80093-3. [DOI] [PubMed] [Google Scholar]
  9. Marinus M. G., Morris N. R. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 1973 Jun;114(3):1143–1150. doi: 10.1128/jb.114.3.1143-1150.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. May M. S., Hattaman S. Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes. J Bacteriol. 1975 Apr;122(1):129–138. doi: 10.1128/jb.122.1.129-138.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. May M. S., Hattman S. Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes. J Bacteriol. 1975 Aug;123(2):768–770. doi: 10.1128/jb.123.2.768-770.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schlagman S., Hattman S., May M. S., Berger L. 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). J Bacteriol. 1976 May;126(2):990–996. doi: 10.1128/jb.126.2.990-996.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Schlagman S., Hattman S. Mutants of the N-3 R-factor conditionally defective in hspII modification and deoxyribonucleic acid-cytosine methylase activity. J Bacteriol. 1974 Oct;120(1):234–239. doi: 10.1128/jb.120.1.234-239.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Vovis G. F., Horiuchi K., Zinder N. D. Endonuclease R-EcoRII restriction of bacteriophage f1 DNA in vitro: ordering of genes V and VII, location of an RNA promotor for gene VIII. J Virol. 1975 Sep;16(3):674–684. doi: 10.1128/jvi.16.3.674-684.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Watanabe T., Takano T., Arai T., Nishida H., Sato S. Episome-mediated Transfer of Drug Resistance in Enterobacteriaceae X. Restriction and Modification of Phages by fi R Factors. J Bacteriol. 1966 Aug;92(2):477–486. doi: 10.1128/jb.92.2.477-486.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES