Analysis of a Core Domain in Drosophila DNA Topoisomerase II

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase II␣, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350 -407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350 -407 in human topoisomerase II␣ seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.Human DNA topoisomerase II is a multifunctional and highly complex enzyme that is able to change the topological conformation of DNA in response to different physiological alterations (1-3). Topological changes mediated by the dimeric topoisomerase II enzyme require a strict control of the passage of duplex DNA through the whole subunit interface (4) and through another duplex coordinately cleaved by the enzyme, where a correct interdomain as well as intersubunit communication is fundamental.Topoisomerase II consists of three distinct domains. The N-terminal and central domains are highly conserved among enzymes from different eukaryotic organisms and also share homology to the gyrase B and A subunits, respectively (5-9). The C-terminal domain is dispensable for in vitro catalytic activity and shows no sequence conservation (9 -11). Central to the activity of the enzyme is its ability to bind and hydrolyze ATP as well as to cleave and religate DNA. The active site for ATP hydrolysis is encompassed in the N-terminal domain (12)(13)(14), while that for DNA cleavage/religation is located in the central domain (15).Structural and biochemical data have suggested a rational model for the catalytic mechanism of eukaryotic topoisomerase . According to this model, the two subunits of the enzyme form a heart-shaped ring structure, where the N-terminal domains protrude as a set of jaws functioning as an ATP operated clamp. In the absence of ATP, the enzyme assumes an open conformation with a gate in the N-terminal part of the enzyme. The open state can permit a DNA segment (the socalled G-segment) to ent...

mentioning

confidence: 55%

Communication between the ATPase and Cleavage/Religation Domains of Human Topoisomerase IIα

Bjergbæk¹,

Kingma²,

Nielsen³

et al. 2000

“…In addition to stabilizing the closed clamp form of topoisomerase II, bisdioxopiperazines inhibit the ATP hydrolysis activity of the eukaryotic enzymes (17,21). In studies with N-terminal fragments of human topoisomerase II␣, bisdioxopiperazines inhibited the ATP hydrolysis activity of these truncated proteins, although the extent of inhibition was less than that observed with the full-length protein, suggesting that protein domains outside of the N terminus influence bisdioxopiperazine inhibition (22,23).…”

mentioning

confidence: 93%

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

Vaughn

Huang

Wessel

et al. 2005

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.

“…Filter binding assays were carried out as described previously (3). Salt-stable closed clamp formation was also determined by analytical ultracentrifugation as described previously (35,37). Briefly, 6 g of an 8.6-kb plasmid was incubated at 37°C with 6 g of human topoisomerase II ␣ or D48N mutant protein in a 40-l reaction mixture containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 100 mM NaCl, 10 mM MgCl 2 , 0.1 mM EDTA, and 50 g/ml bovine serum albumin.…”

Section: Methodsmentioning

confidence: 99%

A Mutation in Human Topoisomerase II α Whose Expression Is Lethal in DNA Repair-deficient Yeast Cells

Walker

Nitiss

Jensen

et al. 2004

Self Cite

Type II DNA topoisomerases are ATP-dependent enzymes that catalyze alterations in DNA topology. These enzymes are important targets of a variety of anti-bacterial and anti-cancer agents. We identified a mutation in human topoisomerase II ␣, changing aspartic acid 48 to asparagine, that has the unique property of failing to transform yeast cells deficient in recombinational repair. In repair-proficient yeast strains, the Asp-48 3 Asn mutant can be expressed and complements a temperature-sensitive top2 mutation. Purified Asp-48 3 Asn Top2␣ has relaxation and decatenation activity similar to the wild type enzyme, but the purified protein exhibits several biochemical alterations compared with the wild type enzyme. The mutant enzyme binds both covalently closed and linear DNA with greater avidity than the wild type enzyme. hTop2␣(Asp-48 3 Asn) also exhibited elevated levels of drug-independent cleavage compared with the wild type enzyme. The enzyme did not show altered sensitivity to bisdioxopiperazines nor did it form stable closed clamps in the absence of ATP, although the enzyme did form elevated levels of closed clamps in the presence of a non-hydrolyzable ATP analog compared with the wild type enzyme. We suggest that the lethality exhibited by the mutant is likely because of its enhanced drug-independent cleavage, and we propose that alterations in the ATP binding domain of the enzyme are capable of altering the interactions of the enzyme with DNA. This mutant enzyme also serves as a new model for understanding the action of drugs targeting topoisomerase II.Type II topoisomerases catalyze changes in DNA topology in an ATP-dependent mechanism (for recent reviews see Refs. 1 and 2). Changes in DNA topology require cleavage of the DNA backbone. For eukaryotic type II topoisomerases, DNA cleavage results from a transesterification reaction between phosphates in the DNA backbone and catalytic tyrosines from each of the two identical subunits of the enzyme, resulting in a protein-bridged DNA double-strand break. ATP binding and hydrolysis are required for the enzyme to carry out catalytic changes in DNA topology, although the high energy cofactor does not play a direct role in the breakage/reunion reaction of the enzyme. Current models suggest that ATP hydrolysis is required for regeneration of the enzyme to complete the catalytic cycle (3, 4), although it has been suggested that ATP hydrolysis may also accelerate strand transfer reactions (5). In addition to an absolute requirement for type II topoisomerase activity in processes such as chromosome segregation (6, 7), these enzymes are targeted by a number of antibacterial and anticancer agents in widespread clinical use. Drugs targeting DNA topoisomerases principally act by increasing the levels of the covalent complex that is an intermediate of the enzyme reaction (reviewed in Refs. 2, 8, and 9).The overall domain organization of prokaryotic and eukaryotic type II topoisomerases is similar, although the eukaryotic enzyme is a homodimer, and the prokaryotic enzymes a...