19F NMR Studies of Vaccinia Type IB Topoisomerase: CONFORMATIONAL DYNAMICS OF THE BOUND DNA SUBSTRATE

Keehwan Kwon,Yu Lin Jiang,Fenhong Song,James T Stivers

doi:10.1074/jbc.m109450200

Abstract

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.

Highlights

The site-specific DNA cleavage and religation activities of the vaccinia virus type IB topoisomerase at (C/T)CCTT؉1X؊1 sites in duplex DNA have allowed detailed investigations of the chemical and conformational steps on the reaction pathway of this enzyme
Chemical probing of the accessibility of all the thymine bases in the DNA substrate using this classic reagent revealed that the ϩ1T of the consensus sequence CCCTTϩ1 was selectively sensitive to oxidation in the phosphotyrosine covalent complex but remained insensitive to this reagent in the free DNA and noncovalent complex with the Y274F mutant
Binding and Kinetic Studies Using ϩ1 UF-containing DNA— Before embarking on NMR studies of the DNA substrate containing 5-F-dUrd, we investigated whether the incorporation of this thymidine analogue resulted in gross changes in the kinetic parameters of the enzyme

Summary

CONFORMATIONAL DYNAMICS OF THE BOUND DNA SUBSTRATE*

In previous kinetic studies of plasmid supercoil relaxation by the vaccinia type I topoisomerase (topo I), it was found that the enzyme removed an average of 5 supercoils during the lifetime of the covalent phosphotyrosine adduct before covalently resealing the DNA phosphodiester backbone [3] This result suggested that the enzyme did not tightly grasp the DNA. The ϩ1T was replaced with the NMR active structural isomer, 5-fluoro-2Ј-deoxyuridine (5-FdUrd) This fluorine-substituted thymine analogue has only a small effect on the thermodynamic and kinetic parameters of the enzyme but provides a useful site-specific probe of the base dynamics at the ϩ1 position. The data suggest that topisomerase I extrudes the ϩ1 base in a dynamic step that precedes DNA cleavage Quantification of these NMR results provides the equilibrium distribution of these DNA conformations in the noncovalent. A model that accommodates these results within the context of the kinetic framework in the accompanying article [7] is presented

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

Substrate and topo