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-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.PVU:II with DNA containing the physiologically relevant N4-methylcytosine modification. R.PVU:II binds (N4m)C-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.PVU:II-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 (N4m)C-modified DNA.
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Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA.
Blumenthal
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RM
D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C039052:N(4)-methylcytosine; D003596:Cytosine; D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific