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Nucleic Acids Research, 2000, Vol. 28, No. 16 3143-3150
© 2000 Oxford University Press

Recognition of native DNA methylation by the PvuII restriction endonuclease

Manda R. Rice and Robert M. Blumenthal*

Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA

Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes. Restriction–modification systems have to distinguish between ‘self’ and ‘non-self’ DNA and depend on the inability of restriction endonucleases to cleave their DNA substrates when the DNA is appropriately methylated. These endonucleases thus provide a model system for studying the recognition of DNA methylation by proteins. We have characterized the interaction of R·PvuII with DNA containing the physiologically relevant N4-methylcytosine modification. R·PvuII binds N4mC-modified DNA and cleaves it very slowly. Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully methylated duplexes, in parallel with a higher binding affinity for hemimethylated DNA. The co-crystal structures of R·PvuII–DNA, together with a mutagenesis study, have implicated specific amino acids in recognition of the methylatable base; one of these is His84. We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slightly increased the cleavage of N4mC-modified DNA.

* To whom correspondence should be addressed. Tel: +1 419 383 5422; Fax: +1 419 383 3002; Email: rblumenthal@mco.edu


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