Substrate recognition by the Pvull endonuclease: binding and cleavage of CAG(5m)CTG sites

The PvuII restriction endonuclease (R.PvuII) cleaves CAG down arrow CTG seq uences as indicated, leaving blunt ends. Its cognate methyltransferase (M.P vuII) generates N4-methylcytosine, yielding CAG(N4m)CTG, though the mechani sm by which this prevents cleavage by R.PvuII is unknown. The heterologous 5-methylcytosine methylation CAG(5m)CTG has also been reported to prevent c leavage by R.PvuII and this has been used in some cloning methods. Since th is heterologous methylation occurs at the native methylated base, it can pr ovide insights into the detection of DNA methylation by R.PvuII. We found t hat the cloned gene for R.PvuII could not stably transform cells protected only by M.AluI (AG(5m)CT) and then determined that R.PvuII cleaves CAG(5m)C TG in vitro, even when both strands are methylated. DNase I footprint analy sis and competition experiments reveal that R.PvuII binds to CA6(5m)CTG spe cifically, though with reduced affinity relative to the unmethylated sequen ce, These results provide biochemical support for the published structures of R.PvuII complexed with DNA containing CAGCTG and CAG(5-iodo)CTG and supp ort a model for how methylation interferes with DNA cleavage by this enzyme .