Interdomain communication in DNA topoisomerase II. DNA binding and enzyme activation.
Topoisomerase II catalyzes the ATP-dependent transport of a DNA segment (T-DNA) through a transient double strand break in another DNA segment (G-DNA). A fundamental mechanistic question is how the individual steps in this process are coordinated. We probed communication between the DNA binding sites and the individual enzymatic activities, ATP hydrolysis, and DNA cleavage. We employed short DNA duplexes to control occupancy at the two binding sites of wild-type enzyme and a variant with a G-DNA site mutation. The DNA concentration dependence of ATP hydrolysis and a fluorescence anisotropy assay provided thermodynamic information about DNA binding. The results suggest that G-DNA binds with higher affinity than T-DNA. Enzyme with only G-DNA bound is competent to cleave DNA, indicating that T-DNA is dispensable for DNA cleavage. The ATPase activity of enzyme bound solely to G-DNA is partially stimulated. Full stimulation requires binding of T-DNA. Both DNA binding sites therefore signal to the ATPase domains. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future mechanistic dissection.
Mueller-Planitz
Felix
F
Department of Biochemistry, School of Medicine, Stanford University, Stanford, California 94305-5307, USA.
Herschlag
Daniel
D
D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006868:Hydrolysis; D009154:Mutation; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II