The DNA Dependence of the ATPase Activity of Human DNA Topoisomerase IIα

Type II DNA topoisomerases catalyze changes in DNA topology and use nucleotide binding and hydrolysis to control conformational changes required for the enzyme reaction. We examined the ATP hydrolysis activity of a bisdioxopiperazine-resistant mutant of human topoisomerase II␣ with phenylalanine substituted for tyrosine at residue 50 in the ATP hydrolysis domain of the enzyme. This substitution reduced the DNA-dependent ATP hydrolysis activity of the mutant protein without affecting the relaxation activity of the enzyme. A similar but stronger effect was seen when the homologous mutation (Tyr 28 3 Phe) was introduced in yeast Top2. The ATPase activities of human TOP2␣(Tyr 50 3 Phe) and yeast Top2(Tyr 28 3 Phe) were resistant to both bisdioxopiperazines and the ATPase inhibitor sodium orthovanadate. Like bisdioxopiperazines, vanadate traps the enzyme in a salt-stable closed conformation termed the closed clamp, which can be detected in the presence of circular DNA substrates. Consistent with the vanadate-resistant ATPase activity, salt-stable closed clamps were not detected in reactions containing the yeast or human mutant protein, vanadate, and ATP. Similarly, ADP trapped wild-type topoisomerase II as a closed clamp, but could not trap either the human or yeast mutant enzymes. Our results demonstrate that bisdioxopiperazine-resistant mutants exhibit a difference in the stability of the closed clamp formed by the enzyme and that this difference in stability may lead to a loss of DNA-stimulated ATPase. We suggest that the DNA-stimulated ATPase of topoisomerase II is intimately connected with steps that occur while the N-terminal domain of the enzyme is dimerized.

Section: Stimulationsupporting

confidence: 82%

“…Gels were then stained with ethidium bromide, and nucleic acids were visualized under UV light. (22,(33)(34)(35). The extent of stimulation depends on both the DNA concentration and the source of topoisomerase II utilized (36).…”

Section: Assays For Closed Clamp Formation Bymentioning

confidence: 99%

Stability of the Topoisomerase II Closed Clamp Conformation May Influence DNA-stimulated ATP Hydrolysis

Vaughn

Huang

Wessel

et al. 2005

“…Enzyme Assays-ATPase assays were performed as described previously (17,18). DNA supercoiling and quinolone-induced cleavage reactions were carried out under the conditions previously described (19).…”

Section: Methodsmentioning

confidence: 99%

“…The 147-, 90-, and 80-bp fragments containing the major gyrase cleavage site of plasmid pBR322 (16) were prepared by polymerase chain reaction using pBR322 as a template. The 40-and 20-bp fragments, based on the same pBR322 site, were prepared by the annealing of complementary chemically synthesized oligonucleotides that had been purified by high performance liquid chromatography (PNACL, University of Leicester).Enzyme Assays-ATPase assays were performed as described previously (17,18). DNA supercoiling and quinolone-induced cleavage reactions were carried out under the conditions previously described (19).…”

mentioning

confidence: 99%

The DNA Gyrase-Quinolone Complex

Kampranis¹,

Maxwell

1998

Self Cite

110

Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606 -22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.DNA gyrase is the intracellular target of the quinolone group of antibacterial agents. The addition of quinolone drugs to an in vitro reaction containing DNA gyrase, relaxed closed-circular DNA, and ATP leads to the inhibition of supercoiling (2). If this reaction is terminated by the addition of a protein denaturant, like SDS, the DNA is found to be cleaved in both strands with a gyrase A subunit (GyrA) 1 covalently attached to each 5Ј-phosphoryl terminus (3). Quinolones also inhibit the relaxation, catenation, and decatenation reactions of gyrase (2, 4).The molecular details of the interaction of the drugs with the gyrase-DNA complex are unknown. When the binding of the drugs to the enzyme was investigated, insignificant binding of quinolones to the A or B subunit (GyrB) or to the gyrase holoenzyme (A 2 B 2 ) was detected (5-7). Binding of the drugs to the DNA is weak, and more efficient in the case of singlecompared with double-stranded DNA (8,9). Quinolones were found to bind strongly to the complex formed by gyrase and DNA, and ATP appeared to assist this interaction (5). These results led to the proposal that the drugs bind to the singlestranded DNA region that is revealed when gyrase cleaves DNA during turnover (10). This proposal was addressed by site-directed mutagenesis of the gyrase active site. Mutation of tyrosine 122 to serine or phenylalanine abolished supercoiling and quinolone-induced DNA cleavage but the mutants could still bind the drugs equally well as wild-type (11). Clearly DNA cleavage is not required for drug binding. Similar results have also been obtained with topoisomerase IV (12).Using limited proteolysis we found that quinolones stabilize a conformational ...

“…Limited proteolysis experiments were performed as described previously (8). ATPase assays were performed as described previously (7,15), in 35 mM Tris⅐HCl (pH 7.5), 24 mM KCl, 4 mM MgCl 2 , 5 mM dithiothreitol, 6.5% w/v glycerol, 2 mM ATP at 25°C. The extent of supercoiling was determined by removing samples from the ATPase reactions and analyzing them by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Hydrolysis of ATP at Only One GyrB Subunit Is Sufficient to Promote Supercoiling by DNA Gyrase

Kampranis

Maxwell

1998

Self Cite

Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala 42 mutant B protein were formed, and the ability of such complexes to hydrolyze ATP was investigated. We found that ATP hydrolysis was able to proceed independently only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolyzed at a time, gyrase could still perform supercoiling, but the limit of this reaction was lower than that observed when both subunits can hydrolyze the nucleotide.The three-dimensional structure of DNA plays a key role in many biological processes. Reactions such as replication, transcription, or recombination not only are regulated by but also have a profound effect on the topology of the DNA molecule. The enzymes responsible for maintaining the topological state of DNA are DNA topoisomerases. One such enzyme is DNA gyrase, a bacterial topoisomerase that introduces negative supercoils into DNA in a reaction coupled to ATP hydrolysis. The action of gyrase involves the creation of a double-stranded break in one DNA segment and the passage of another segment through this enzyme-stabilized DNA gate.The ability of gyrase to negatively supercoil DNA is unique among topoisomerases and is based on its mode of DNA binding (1, 2). Gyrase wraps DNA in a right-handed manner (1), resulting in the positioning of two segments of DNA in the right orientation for supercoiling. Binding of ATP closes a protein clamp that traps the DNA segment to be transported. The nucleotide is then hydrolyzed, and the free energy is coupled to the supercoiling reaction. After hydrolysis, the enzyme is reset for another round of supercoiling. The limit of supercoiling is believed to be thermodynamic rather than steric, because gyrase can supercoil very small DNA circles, whereas a nucleotide analog (ATP␣S) 1 with higher free energy of hydrolysis than ATP is capable of taking the limit of the supercoiling reaction to higher negative superhelical density (3,4).Gyrase is a heterotetramer in which two A (GyrA, 97 kDa) and two B (GyrB, 90 kDa) subunits constitute an A 2 B 2 complex (5). There is one ATP-binding site per GyrB, which is situated in the 43-kDa N-terminal domain of the protein. The structure of this domain complexed with the ATP analog 5Ј-adenylyl-␤,␥-imidodiphosphate (ADPNP) was solved by x-ray crystallography and was found to be a dimer (6). Study of the ATPase reaction of this domain revealed that dimerization is an essential step for ATP hydrolysis (7). It is very likely that dimerization of the 43-kDa domain also occurs in the ATPase reaction of intact gyrase (8). The rate of ATP hydrolysis by gyrase is stimulated by the presence of DNA (9), and the kinetics of hydrolysis show positive cooperativity between the two ATPbinding sites (10 -12).A number of issues concerning the mechanism of ATP hydrolysis are still unclear. These include the mechanism of cooperativity between the two sites and the coupling of the free ener...