DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment. In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport. Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction. Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner. This result is incongruous with the models based on the nonhydrolyzable ATP analog data. Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport. These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover. A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.T ype II DNA topoisomerases are ubiquitous enzymes essential for the unlinking of intertwined chromosomes, chromosomal condensation͞decondensation, and manipulation of DNA supercoiling (1, 2). These enzymes catalyze the ATP-dependent transport of one segment of DNA through a transient, enzymemediated break in a second DNA segment (for recent reviews, see refs. 3 and 4). The eukaryotic topoisomerase II enzymes are homodimers, with a monomer molecular mass of Ϸ160-180 kDa, making them relatively simple macromolecular machines for studying the coupling of ATP usage to complex protein movements. The N-terminal half of the enzyme is homologous to Escherichia coli gyrase B and contains the ATPase active site; the C-terminal half is homologous to gyrase A and contains the active site tyrosine for DNA cleavage and the primary dimerization interface. In addition to being mechanistically fascinating, the prokaryotic and eukaryotic members of this enzyme family are the targets of numerous antibiotic and anticancer drugs, respectively (5-7).The mechanism of topoisomerase II is known to involve several steps and associated protein conformational changes (reviewed in ref. 4). The enzyme binds a segment of DNA, the gate or G segment. Both strands of this DNA segment are cleaved and religated by a pair of active site tyrosines, one in each monomer of the dimer. The cleavage reaction results in a four-base staggered break in the DNA, with an enzyme monomer covalently attached to each 5Ј phosphate. Upon binding ATP, the amino-terminal ATPase domains of the enzyme dimerize, capturing a second segment of DNA, the transport or T segment, within the enzyme clamp. The ends of the cleaved G segment are separated, and the T segment is transported through the opening. The gate in the G segment is closed. The T segment is transported out of the topoisomerase, most likely through the C-terminal dimerization interface. The amino terminal dimerization interface re...

Regulation of cellular functions can be accomplished by many mechanisms, including transcriptional regulation, alternative splicing, translational regulation, phosphorylation and other posttranslational covalent modifications, degradation, localization, proteinprotein interactions, and small-molecule allosteric effectors. Largely because of advances in the techniques of molecular biology in the past few decades, our knowledge of regulation by most of these mechanisms has expanded enormously. Regulation by smallmolecule, allosteric interactions is an exception. Many of the best-known allosteric regulators were discovered decades ago, when we knew little about all of these other forms of regulation. Allostery is the most direct, rapid, and efficient regulatory mechanism to sense changes in the concentration of small molecules and alter cellular responses to maintain homeostasis. In this perspective, we present the argument that allosteric regulation is underappreciated in the systems biology world and that many allosteric effectors remain to be discovered.

In the preceding paper, we showed that DNA topoisomerase II from Saccharomyces cerevisiae binds two ATP and rapidly hydrolyzes at least one of them before encountering a slow step in the reaction mechanism. These data are potentially consistent with two different types of reaction pathways: (1) sequential ATP hydrolysis or (2) simultaneous hydrolysis of both ATP. Here, we present results that are consistent only with topoisomerase II hydrolyzing its two bound ATP sequentially. Additionally, these results indicate that the products of the first hydrolysis are released from the enzyme before the second ATP is hydrolyzed. Release of products from both the first and second hydrolyses contributes to the rate-determining process. The proposed mechanism for ATP hydrolysis by topoisomerase II is complex, having nine rate constants. To calculate values for each of these rate constants, a technique of kinetic parameter estimation was developed. This technique involved using singular perturbation theory in order to estimate rate constants, and consequently identify kinetic steps following the rate-determining step.

The mechanism by which T7 DNA polymerase (exo-) bypasses N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) adducts was studied by single-turnover kinetics. These adducts are known to be mutagenic in several cell types, and their bypass was studied in the framework of understanding how they promote mutations. Synthetic primer/templates were made from a template sequence containing a single guanine, to which the adducts were covalently attached, and one of three primers whose 3' ends were various distances from the adduct in the annealed substrates. Upon approaching the site of either adduct, the polymerase was found to add nucleotides as rapidly as to unmodified primer/templates, until just opposite the lesion. The incorporation rate of dCTP (at 100 microM) opposite AF-dG or AAF-dG was approximately 5 x 10(4)- and 4 x 10(6)-fold slower, respectively, than incorporation at the same position into an unmodified primer/template. The polymerase dissociated from the sites of the adducts at approximately the same rate that it dissociated from unmodified DNA. Correct nucleotide incorporation was favored both opposite and immediately after AF-dG. However, at both positions, dATP was the most rapidly misincorporated nucleotide. Misincorporation of dATP was more rapid than correct nucleotide incorporation both opposite and immediately after AAF-dG. These results are discussed in terms of the effects of AF and AAF adducts in vivo.

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