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.