CATALYTIC AND DNA-BINDING PROPERTIES OF P-UPSILON-UII RESTRICTION-ENDONUCLEASE MUTANTS

The role of particular residues of the PvuII endonuclease in DNA bindi ng and cleavage was studied by mutational analysis using a number of i n vivo and in vitro approaches. While confirming the importance of res idues predicted to be involved directly in function by the crystal str ucture, the analysis led to several striking results. Aspartate 34, wh ich contacts the central base pair of the PvuII site (5'-CAGCTG-3') th rough the minor groove, plays a critical role in binding specificity. A D34G mutant binds with high affinity to any of the sequences in the set CANNTG, although its low level of cleavage activity acts only on t he wild-type site. In addition, a His to Ala mutation at the residue t hat contacts the central G and is predicted to be blocked by PvuII met hylation still requires the PvuII methylase to be maintained in vivo, arguing against this hypothesis as the only mechanism for methylation protection. Finally, four of the five mutations that reduce cleavage a ctivity while still exhibiting binding in the gel shift assay are at r esidues that form DNA- or subunit-subunit contacts rather than in the catalytic center. This provides further evidence for a strong linkage between specific binding and catalysis.