Coupling between ATP binding and DNA cleavage by DNA topoisomerase II: A unifying kinetic and structural mechanism.
DNA topoisomerase II is a molecular machine that couples ATP hydrolysis to the transport of one DNA segment through a transient break in another segment. To learn about the energetic connectivity that underlies this coupling, we investigated how the ATPase domains exert control over DNA cleavage. We dissected the DNA cleavage reaction by measuring rate and equilibrium constants for the individual reaction steps utilizing defined DNA duplexes in the presence and absence of the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMPPNP). Our results revealed the existence of two enzyme conformations whose relative abundance is sensitive to the presence of nucleotides. The predominant species in the absence of nucleotides binds DNA at a diffusion limited rate but cannot efficiently cleave DNA. In the presence of AMPPNP, most of the enzyme is converted to a state in which DNA binding and release is extremely slow but which allows DNA cleavage. A minimal kinetic and thermodynamic framework is established that accounts for the cooperativity of cleavage of the two DNA strands in the presence and absence of bound AMPPNP and includes conformational steps revealed in the kinetic studies. The model unifies available kinetic, thermodynamic, and structural data to provide a description for the reaction in terms of the order and rate of individual reaction steps and the physical nature of the species on the reaction path. Furthermore, this reaction framework provides a foundation for a future in-depth analysis of energy transduction by topoisomerase II, for guiding and interpreting future structural studies, and for analyzing the mechanism of drugs that convert topoisomerase into a cellular poison.
Mueller-Planitz
Felix
F
Department of Biochemistry, School of Medicine, Stanford University, Stanford, California 94305, USA.
Herschlag
Daniel
D
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D016880:Anisotropy; D002384:Catalysis; D004247:DNA / Q000737:chemistry*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors
D009711:Nucleotides; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004250:DNA Topoisomerases, Type II