Recognition of native DNA methylation by the PvuII restriction endonuclease

AUTOR(ES)

Rice, Manda R.

FONTE

Oxford University Press

RESUMO

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.

Documentos Relacionados

• The inhibition of restriction endonuclease PvuII cleavage activity by methylation outside its recognition sequence.
• Alteration of the specificity of PvuII restriction endonuclease.
• Structure of PvuII endonuclease with cognate DNA.
• Sequence and characterization of pvuIIR, the PvuII endonuclease gene, and of pvuIIC, its regulatory gene.
• Host-mediated modification of PvuII restriction in Mycobacterium tuberculosis.