Abstract
Eukaryotic type IB topoisomerases catalyze the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The 314-amino acid vaccinia topoisomerase is the smallest member of this family and is distinguished from its cellular counterparts by its specificity for cleavage at the target sequence 5'-CCCTT downward arrow. Here we show that Topo-(81-314), a truncated derivative that lacks the N-terminal domain, performs the same repertoire of reactions as the full-sized topoisomerase: relaxation of supercoiled DNA, site-specific DNA transesterification, and DNA strand transfer. Elimination of the N-terminal domain slows the rate of single-turnover DNA cleavage by 10(-3.6), but has little effect on the rate of single-turnover DNA religation. DNA relaxation and strand cleavage by Topo-(81-314) are inhibited by salt and magnesium; these effects are indicative of reduced affinity in noncovalent DNA binding. We report that identical properties are displayed by a full-length mutant protein, Topo(Y70A/Y72A), which lacks two tyrosine side chains within the N-terminal domain that contact the DNA target site in the major groove. We speculate that Topo-(81-314) is fully competent for transesterification chemistry, but is compromised with respect to a rate-limiting precleavage conformational step that is contingent on DNA contacts made by Tyr-70 and Tyr-72.
Highlights
The 314-amino acid vaccinia enzyme is the smallest topoisomerase known and affords a more tractable target for structure-function studies than the cellular type IB enzymes, which range from 765 to 1019 amino acids (2, 3)
Relaxation of Supercoiled DNA by Topo-(81–314)—Topo-(81– 314) is a truncated version of vaccinia topoisomerase that lacks the N-terminal structural domain defined by limited proteolysis with trypsin (Fig. 1)
The results presented in this study of vaccinia topoisomerase enhance our understanding of the eukaryotic type IB enzyme family as follows. (i) They define a catalytically active domain that is conserved between the cellular and poxvirus enzymes; (ii) they illuminate a clear distinction between structural elements required for transesterification chemistry in general and those required for the cleavage reaction; (iii) they demonstrate that the catalytic domain per se is capable of recognizing the target sequence for transesterification
Summary
The 314-amino acid vaccinia enzyme is the smallest topoisomerase known and affords a more tractable target for structure-function studies than the cellular type IB enzymes, which range from 765 to 1019 amino acids (2, 3). Specific functional groups identified through mutagenesis as being required for transesterification chemistry are situated near the hinge and within the C-terminal domain These include the active site nucleophile (Tyr-274) and four other residues (Arg-130, Lys-167, Arg223, and His-265) that are essential for the DNA cleavage and religation steps (11–15, 17, 19). Mutational effects on DNA binding are evinced by inhibition of topoisomerase activity in the presence of magnesium and salt (16, 18) These results have prompted the suggestion (9) that low affinity DNA binding and reaction chemistry are performed by the carboxyl two-thirds of the vaccinia enzyme, the sequence of which is similar to that of the cellular topoisomerases, whereas discrimination of the DNA sequence at the cleavage site is facilitated by the N-terminal domain, which is divergent in sequence and three-dimensional structure between the viral and cellular enzymes (20, 21). We propose a revised model for catalysis whereby the N-terminal domain enhances DNA binding and is required for a pre-cleavage conformational step
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