Abstract
Eukaryotic type 1B topoisomerases act by forming covalent enzyme-DNA intermediates that transiently nick DNA and thereby release DNA supercoils. Here we present a study of the topoisomerase encoded by the pathogenic poxvirus molluscum contagiosum. Our studies of DNA sites favored for catalysis reveal a larger recognition site than the 5'-(T/C)CCTT-3' sequence previously identified for poxvirus topoisomerases. Separate assays of initial DNA binding and covalent complex formation revealed that different DNA sequences were important for each reaction step. The location of the protein-DNA contacts was mapped by analyzing mutant sites and inosine-substituted DNAs. Some of the bases flanking the 5'-(T/C)CCTT-3' sequence were selectively important for covalent complex formation but not initial DNA binding. Interactions important for catalysis were probed with 5'-bridging phosphorothiolates at the site of strand cleavage, which permitted covalent complex formation but prevented subsequent religation. Kinetic studies revealed that the flanking sequences that promoted recovery of covalent complexes increased initial cleavage instead of inhibiting resealing of the nicked intermediate. These data 1) indicate that previously unidentified DNA contacts can accelerate a step between initial binding and covalent complex formation and 2) help specify models for conformational changes promoting catalysis.
Highlights
Topoisomerases play important roles in DNA replication, recombination, and transcription [1]
Each contained the 5Ј-(T/ C)CCTT-3Ј sequence favored by poxvirus topoisomerases [2, 10]
Our studies indicated that MCV topoisomerase recognizes a larger site than the previously described 5Ј-(C/T)CCTT-3Ј motif
Summary
Enzyme Purification—MCV topoisomerase was overexpressed in Escherichia coli and purified as described by Hwang et al [7]. Reaction mixture contained 20 mM Tris-Cl (pH 8.0), 200 mM potassium glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM EDTA, and 50 nM of 5Ј-32P-end-labeled DNA substrate. Tris-Cl (pH 8.0), 200 mM potassium-glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM EDTA, 50 nM of 5Ј-32P-end-labeled DNA substrate and 20 nM topoisomerase. Reaction products were heated at 95 °C for 5 min and analyzed on 12% polyacrylamide gels containing 0.1% SDS. Reaction mixtures (10 l) contained 20 mM Tris-Cl (pH 8.0), 200 mM potassium glutamate, 1 mM dithiothreitol, 0.1% Triton X-100, 1 mM EDTA, 50 nM of 5Ј-32P-end-labeled DNA substrate, and topoisomerase. Reaction mixtures were prewarmed at 37 °C, initiated by the addition of topoisomerase, and incubated for 5 min at 37 °C. One-l samples were analyzed on 10% DNA sequencing-type polyacrylamide gels and analyzed by autoradiography
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