In Vivo Methylation of Bacteriophage φX174 DNA

Author:

Hattman Stanley1,Gribbin Chris1,Hutchison Clyde A.2

Affiliation:

1. Department of Biology, University of Rochester, Rochester, New York 14627

2. Department of Bacteriology, University of North Carolina, Chapel Hill, North Carolina 27514

Abstract

A mutant (designated mec ) has been isolated from Escherichia coli C which has lost DNA-cytosine methylase activity and the ability to protect phage λ against in vivo restriction by the RII endonuclease. This situation is analogous to that observed with an E. coli K-12 mec mutant; thus, the E. coli C methylase appears to have overlapping sequence specificity with the K-12 and RII enzymes; (the latter methylases have been shown previously to recognize the same sequence). Covalently closed, supertwisted double-standed DNA (RFI) was isolated from C mec + and C mec cells infected with bacteriophage φX174. φX· mec RFI is sensitive to in vitro cleavage by R· Eco RII and is cut twice to produce two fragments of almost equal size. In contrast, φX· mec + RFI is relatively resistant to in vitro cleavage by R· Eco RII. R· Bst I, which cleaves mec + /RII sites independent of the presence or absence of 5-methylcytosine, cleaves both forms of the RFI and produces two fragments similar in size to those observed with R· Eco RII. These results demonstrate that φX· mec + RFI is methylated in vivo by the host mec + enzyme and that this methylation protects the DNA against cleavage by R· Eco RII. This is consistent with the known location of two mec + / RII sequences (viz., [Formula: see text]) on the φX174 map. Mature singlestranded virion DNA was isolated from φX174 propagated in C mec + or C mec in the presence of l -[ methyl - 3 H]methionine. Paper chromatographic analyses of acid hydrolysates revealed that φX· mec + DNA had a 10-fold-higher ratio of [ 3 H]5-methylcytosine to [ 3 H]cytosine compared to φX· mec . Since φX· mec + contains, on the average, approximately 1 5-methylcytosine residue per viral DNA, we conclude that methylation of φX174 is mediated by the host mec + enzyme only. These results are not consistent with the conclusions of previous reports that φX174 methylation is mediated by a phage-induced enzyme and that methylation is essential for normal phage development.

Publisher

American Society for Microbiology

Subject

Virology,Insect Science,Immunology,Microbiology

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