Recognition of native DNA methylation by the PvuII restriction endonuclease

Recognizing the methylation status of specific DNA sequences is central to the function of many systems in eukaryotes and prokaryotes, Restriction-mod ification systems have to distinguish between 'self' and 'non-self' DNA and depend on the inability of restriction endonucleases to cleave their DNA s ubstrates when the DNA is appropriately methylated. These endonucleases thu s provide a model system for studying the recognition of DNA methylation by proteins, We have characterized the interaction of R . PvuII with DNA cont aining the physiologically relevant N4-methylcytosine modification. R . Pvu II binds C-N4m-modified DNA and cleaves it very slowly, Methylated strands in hemimethylated duplexes were cleaved at a higher rate than in fully meth ylated duplexes, in parallel with a higher binding affinity for hemimethyla ted DNA, The co-crystal structures of R . PvuII-DNA, together with a mutage nesis study, have implicated specific amino acids in recognition of the met hylatable base; one of these is His84. We report that replacing His84 with Ala reduced the rate of cleavage of unmodified DNA but, in contrast, slight ly increased the cleavage of C-N4m-modified DNA.