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Updated: Aug 14, 2026
Methylated DNA Immunoprecipitation
Published on: January 2, 2009
Recognition of native DNA methylation by the PvuII restriction endonuclease
1Department of Microbiology and Immunology, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA.
Abstract:
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.
Insights
Restriction-modification enzymes like R.PVU:II recognize DNA methylation. His84 is key for this, as mutations affect cleavage rates of modified and unmodified DNA.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA methylation is crucial for cellular processes in prokaryotes and eukaryotes.
- Restriction-modification systems use DNA methylation to differentiate self from non-self DNA.
- Restriction endonucleases serve as models for studying protein-DNA methylation interactions.
Purpose of the Study:
- To characterize the interaction of R.PVU:II with N4-methylcytosine modified DNA.
- To investigate the role of specific amino acids, particularly His84, in DNA methylation recognition by R.PVU:II.
Main Methods:
- Biochemical characterization of R.PVU:II activity on modified DNA substrates.
- Analysis of R.PVU:II binding affinity and cleavage rates for hemimethylated and fully methylated DNA.
- Site-directed mutagenesis of R.PVU:II, focusing on His84, followed by enzymatic assays.
- Co-crystal structure determination of R.PVU:II-DNA complexes.
Main Results:
- R.PVU:II binds and very slowly cleaves N4-methylcytosine modified DNA.
- Cleavage rates and binding affinity were higher for hemimethylated DNA compared to fully methylated DNA.
- Mutating His84 to Ala decreased cleavage of unmodified DNA but increased cleavage of N4-methylcytosine modified DNA.
Conclusions:
- His84 is critical for R.PVU:II's recognition of DNA methylation.
- The enzyme exhibits differential activity towards hemimethylated versus fully methylated DNA.
- Understanding these interactions provides insights into DNA methylation recognition mechanisms.
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