title: Fluorescence spectroscopy studies of vaccinia type IB DNA topoisomerase. Closing of the enzyme clamp is faster than DNA cleavage.
abstract: The prototypic type IB topoisomerase isolated from vaccinia virus cleaves the phosphodiester backbone of duplex DNA at the sequence 5'-(C/T)CCTT, forming a covalent 3'-phosphotyrosyl adduct. A precleavage conformational change in which the enzyme clamps circumferentially around the DNA has been implicated on the basis of structural and biochemical studies. However, no direct measurements to elucidate this key step have been obtained to date. To address this shortcoming we have developed two new fluorescence assays that allow detection of conformational changes in both the enzyme and substrate DNA, and allow determination of the thermodynamic and kinetic mechanism for noncovalent DNA binding and phosphodiester cleavage. The results indicate that clamp closing occurs in a rapid step (>25 s(-1)) that is at least 14-fold faster than the maximal rate of DNA cleavage. Opening of the clamp to release the noncovalently bound substrate is also 5-8-fold more rapid than DNA cleavage. We propose a model in which DNA cleavage and religation are connected through a single high energy transition state involving covalent bond breaking. Alternative models that involve a slow precleavage conformational step are not easily reconciled with the available data.
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
authors.forename: Keehwan
authors.initials: K
authors.lastname: Kwon
authors.forename: James T
authors.initials: JT
authors.lastname: Stivers
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D012965:Sodium Chloride; D004247:DNA; D004264:DNA Topoisomerases, Type I