title: Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding.
abstract: Previous structural studies on native T5 5' nuclease, a member of the flap endonuclease family of structure-specific nucleases, demonstrated that this enzyme possesses an unusual helical arch mounted on the enzyme's active site. Based on this structure, the protein's surface charge distribution, and biochemical analyses, a model of DNA binding was proposed in which single-stranded DNA threads through the archway. We investigated the kinetic and substrate-binding characteristics of wild-type and mutant nucleases in relation to the proposed model. Five basic residues R33, K215, K241, R172, and R216, are all implicated in binding branched DNA substrates. All these residues except R172 are involved in binding to duplex DNA carrying a 5' overhang. Replacement of either K215 or R216 with a neutral amino acid did not alter kcat appreciably. However, these mutant nucleases displayed significantly increased values for Kd and Km. A comparison of flap endonuclease binding to pseudoY substrates and duplexes with a single-stranded 5' overhang suggests a better model for 5' nuclease-DNA binding. We propose a major revision to the binding model consistent with these biophysical data.
authors.affiliation: Division of Genomic Medicine, University of Sheffield Medical School, Krebs Institute, Beech Hill Road, Sheffield S10 2RX, United Kingdom.
authors.forename: Joe J
authors.initials: JJ
authors.lastname: Dervan
authors.forename: Min
authors.initials: M
authors.lastname: Feng
authors.forename: Dipak
authors.initials: D
authors.lastname: Patel
authors.forename: Jane A
authors.initials: JA
authors.lastname: Grasby
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Artymiuk
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Ceska
authors.forename: Jon R
authors.initials: JR
authors.lastname: Sayers
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004706:Endodeoxyribonucleases