A role for the passage helix in the DNA cleavage reaction of eukaryotic topoisomerase II. A two-site model for enzyme-mediated DNA cleavage.
Eukaryotic topoisomerase II is capable of binding two separate nucleic acid helices prior to its DNA cleavage and strand passage events (Zechiedrich, E. L., and Osheroff, N (1990) EMBO J. 9, 4555-4562). Presumably, one of these helices represents the helix that the enzyme cleaves (i.e. cleavage helix), and the other represents the helix that it passes (i.e. passage helix) through the break in the nucleic acid backbone. To determine whether the passage helix is required for reaction steps that precede the enzyme's DNA strand passage event, interactions between Drosophila melanogaster topoisomerase II and a short double-stranded oligonucleotide were assessed. These studies employed a 40-mer that contained a specific recognition/cleavage site for the enzyme. The sigmoidal DNA concentration dependence that was observed for cleavage of the 40-mer indicated that topoisomerase II had to interact with more than a single oligonucleotide in order for cleavage to take place. Despite this requirement, results of enzyme DNA binding experiments indicated no binding cooperativity for the 40-mer. These findings strongly suggest a two-site model for topoisomerase II action in which the passage and the cleavage helices bind to the enzyme independently, but the passage helix must be present for efficient topoisomerase II-mediated DNA cleavage to occur.
Corbett
A H
AH
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Zechiedrich
E L
EL
Osheroff
N
N
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D004250:DNA Topoisomerases, Type II / Q000378:metabolism* / Q000648:ultrastructure; D004331:Drosophila melanogaster / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D009841:Oligonucleotides; D004247:DNA; D004250:DNA Topoisomerases, Type II