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Biochemistry. 1994 Jan 11;33(1):327-39.

Vaccinia DNA topoisomerase I: single-turnover and steady-state kinetic analysis of the DNA strand cleavage and ligation reactions.

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1
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.

Abstract

Vaccinia DNA topoisomerase I catalyzes a reversible, site-specific strand cleavage and resealing reaction with duplex DNA involving a transient 3'-phosphotyrosyl linkage between the DNA and Tyr-274 of the enzyme. Single-turnover and steady-state kinetic measurements, as well as DNA binding studies with DNA duplexes containing the preferred cleavage sequence (5'-CCCTT decreases -3') in 50 mM Tris-HCl, pH 7.5, at 20 degrees C, have permitted the evaluation of the individual rate constants for strand cleavage (kcl) and religation (kr) and for duplex DNA binding and dissociation. The values of kcl = 0.07 s-1 and kr = 0.66 s-1 indicate that the internal cleavage equilibrium (Kcl = kcl/kr = 0.1) favors the uncleaved E.DNA complex. The apparent second-order rate constant kcl/KmDNA + = 8 x 10(5) M-1 s-1 for the single-turnover cleavage reaction is 10(2)-10(3)-fold less than the rate of diffusional encounter and provides an estimate of k(on) (DNA). Single-turnover cleavage experiments using a duplex substrate with a nonbridging racemic phosphorothioate nucleotide substitution at the cleavage site showed biphasic cleavage kinetics with equal amplitudes for each phase, which was fit to a double exponential: kfast = 0.01 s-1 and kslow = 0.0004 s-1. These "thio effects" (= kclphos/kclthiol) of 4.6- and 115-fold indicate that cleavage is at least partially rate-limiting in the single-turnover reaction; the two kinetic phases indicate a strong preference for cleavage of one thio isomer. Multiple-turnover cleavage-religation reactions showed an initial pre-steady-state burst proportional to enzyme, followed by a slower steady-state rate with a value of kcat = 0.006 s-1. The phosphorothioate substrate showed a smaller burst and no significant thio effect on kcat. These results indicate fast chemical steps and largely rate-limiting dissociation of the religated product (KD = 54 nM) in the steady-state, a conclusion confirmed by direct measurement of the rate constant for product dissociation as 0.01 s-1. MgCl2 (5 mM) increases this rate constant by an order of magnitude, thus explaining the divalent cation induced acceleration of DNA supercoil relaxation by this enzyme. No divalent cation binding by the enzyme was detected indicating this effect to result from metal binding to DNA. The rate constants kcl and kr for the nucleophilic attack of Tyr-274 and deoxyribose 5'-OH at phosphorus represent approximately 10(9)- and 10(12)-fold enhancements over the analogous attack of phenolate and alcoholate anions at the phosphorus backbone of DNA in solution at pH 7.5. Consistent with this estimate, the Y274F mutant is at least 10(6)-fold less active in cleavage than the wild-type enzyme.

PMID:
8286354
[Indexed for MEDLINE]
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