title: Catalytic and DNA binding properties of PvuII restriction endonuclease mutants.
abstract: The role of particular residues of the PvuII endonuclease in DNA binding and cleavage was studied by mutational analysis using a number of in vivo and in vitro approaches. While confirming the importance of residues predicted to be involved directly in function by the crystal structure, the analysis led to several striking results. Aspartate 34, which contacts the central base pair of the PvuII site (5'-CAGCTG-3') through 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 the wild-type site. In addition, a His to Ala mutation at the residue that contacts the central G and is predicted to be blocked by PvuII methylation 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 activity while still exhibiting binding in the gel shift assay are at residues 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.
authors.affiliation: New England Biolabs Incorporated, Beverly, Massachusetts 01915, USA.
authors.forename: H G
authors.initials: HG
authors.lastname: Nastri
authors.forename: P D
authors.initials: PD
authors.lastname: Evans
authors.forename: I H
authors.initials: IH
authors.lastname: Walker
authors.forename: P D
authors.initials: PD
authors.lastname: Riggs
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium