Substrate recognition by the PvuII endonuclease: Binding and cleavage of CAG5mCTG sites

Abstract

The PvuII restriction endonuclease (R·PvuII) cleaves CAG↓CTG sequences as indicated, leaving blunt ends. Its cognate methyltransferase (M·PvuII) generates N4-methylcytosine, yielding CAG(N4m)CTG, though the mechanism by which this prevents cleavage by R·PvuII is unknown. The heterologous 5-methylcytosine methylation CAG5mCTG has also been reported to prevent cleavage by R·PvuII and this has been used in some cloning methods. Since this heterologous methylation occurs at the native methylated base, it can provide insights into the detection of DNA methylation by R·PvuII. We found that the cloned gene for R·PvuII could not stably transform cells protected only by M AluI (AG5mCT) and then determined that R·PvuII cleaves CAG5mCTG in vitro, even when both strands are methylated. DNase I footprint analysis and competition experiments reveal that R·PvuII binds to CAG5mCTG specifically, though with reduced affinity relative to the unmethylated sequence. These results provide biochemical support for the published structures of R·PvuII complexed with DNA containing CAGCTG and CAG(5-iodo)CTG and support a model for how methylation interferes with DNA cleavage by this enzyme.