19F NMR studies of vaccinia type IB topoisomerase. Conformational dynamics of the bound DNA substrate.
The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT(+1)X(-1) sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme (see accompanying article (Kwon, K., and Stivers, J. T. (2002) J. Biol. Chem. 277, 345-352)). To extend these studies to the DNA substrate, we have performed 19F NMR experiments using substrates in which the +1 T has been replaced with the NMR-sensitive thymidine base analogue 5-fluoro-2'-deoxyuridine (5-F-dUrd). Substitution of 5-F-dUrd has little effect on the binding affinity of topoisomerase I for DNA, results in small changes in the cleavage and religation rate constants, and produces a net 3-fold decrease in the cleavage equilibrium constant as compared with the CCCTT consensus DNA. One-dimensional 19F NMR experiments show that the +1 5-F-dUrd is in a dynamic equilibrium between a stacked and unstacked state in both the noncovalent complex and the covalent phosphotyrosine complex. These NMR observations are supported by the selective sensitivity of the +1 T and +1 5-F-dUrd to KMnO4 oxidation. A role for localized DNA distortion in the topoisomerase I mechanism is suggested.
Kwon
Keehwan
K
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
Jiang
Yu Lin
YL
Song
Fenhong
F
Stivers
James T
JT
D004247:DNA / Q000737:chemistry*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D014616:Vaccinia virus / Q000201:enzymology*
D004247:DNA; D004264:DNA Topoisomerases, Type I