Domain communication in DNA topoisomerase II

DNA topoisomerase II is a molecular machine that couples the expenditure of ATP to transport of one DNA segment, the T-DNA, through a transient double strand break in another DNA segment, the G-DNA. A fundamental mechanistic question is how the individual steps in this process are coordinated. To unravel the intramolecuiar communication responsible for coupling we developed methods to physically or temporally isolate different reaction complexes and intermediates and dissected the behavior of these species. We employed short DNA duplexes of defined sequence to control occupancy at the two binding sites and enriched the ATPase domains with different nucleotides and nucleotide analogs. ATP hydrolysis, DNA cleavage and a fluorescence anisotropy assay were employed to thermodynamically and kinetically characterize individual species. The results provide insights into the DNA cleavage site recognition process, into the communication between the ATPase and the DNA cleavage domains and into the communication between the DNA binding sites and the two enzymatic activities. Binding of Gand T-DNA participate in activating the ATP turnover, indicating that the ATPase domains are energetically in contact with both DNA binding sites. The cleavage site in the G-DNA is recognized in a conformational step that takes place after DNA binding, but before the cleavage of the first DNA strand. A kinetic dissection of the DNA cleavage reaction confirms that the enzyme-DNA complex must undergo a conformational change before the DNA strands can be broken. The binding energy of the nucleotides is used to promote this conformational rearrangement. Previous studies have shown that nucleotide binding induces closure of the ATPase domains. Thus, closure of the ATPase domains provides an attractive and simple structural explanation for the communication between the ATPase and the DNA cleavage domains. This model implicates the ATPase domains as strict regulators of DNA cleavage. The results support and extend current mechanistic models for topoisomerase II-catalyzed DNA transport and provide a framework for future in-depth mechanistic dissections of the intramolecular communication of topoisomerase II.