Abstract
Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.
Highlights
Stewart Shuman and John Prescott From the Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021
Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature
Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission
Summary
Stewart Shuman and John Prescott From the Program in Molecular Biology, Sloan-Kettering Institute, New York, New York 10021. Cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr+Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, bound. Camptothecin has been used, to explore the interaction of topoisomerase I with specific DNA elements in vivo (Gilmour and Elgin, 1987; Stewart and Schutz, 1987; Culotta and Sollner-Webb, 1988; Zhang et al, 1988; Porter and Champoux, 1989; Stewart et al, 1990) Such studies support the association, temporally and physically, of topoisomerase I with actively transcribed DNA sequences. Camptothecin appears to shift the equilibria of topoisomerase-DNA interactions toward the covalent intermediate, and does so by binding to the enzyme-DNA complex
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