title: Changing the enzymatic activity of T7 endonuclease by mutations at the beta-bridge site: alteration of substrate specificity profile and metal ion requirements by mutation distant from the catalytic domain.
abstract: Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge. The activities of enzymes with mutations in the beta-bridge segment were studied. Mutations that did not affect catalytic domain folding and function but did alter the relative positions of these domains retained catalytic activity but with altered specificity and metal ion dependence. Our results suggest that the enzyme recognizes its substrates by DNA conformation exclusion and offer a simple explanation for the broad substrate specificity of phage resolvase.
authors.affiliation: New England Biolabs, Inc., 32 Tozer Rd, Beverly, Massachusetts 01915, USA. Guan@neb.com
authors.forename: Chudi
authors.initials: C
authors.lastname: Guan
authors.forename: Sanjay
authors.initials: S
authors.lastname: Kumar
authors.forename: Rebecca
authors.initials: R
authors.lastname: Kucera
authors.forename: Amy
authors.initials: A
authors.lastname: Ewel
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D020137:Base Pair Mismatch / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D008345:Manganese; D003850:Deoxyribonuclease I; D008274:Magnesium