Analysis of Functional Domain Organization in DNA Topoisomerase II from Humans and Saccharomyces cerevisiae

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase II␣ enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase II␣ enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.Type II DNA topoisomerases are highly complex enzymes that are able to change the topological conformation of DNA by introducing a transient double strand break in one DNA helix, the G-segment, whereas another intact helix, the T-segment, is transported coordinately through the gated DNA (1). The process depends on ATP, which drives the enzyme through a series of conformational changes (2-5).The homodimeric eukaryotic topoisomerase II enzyme contains two highly conserved catalytic entities, which also share homology with the bacterial DNA topoisomerase II counterpart, DNA gyrase. The ATPase activity is found in the Nterminal region, whereas the DNA cleavage and ligation activities are held by the central region (6). The extreme C termini of the type II topoisomerases are divergent and dispensable for catalytic activity in vitro (7-9).The structural data reveal that the N-terminal region of topoisomerase II forms an ATP-operated clamp, which closes upon ATP binding (10, 11). The dimeric N-terminal clamp contains two domains in each protomer (10). The most Nterminal domain is responsible for dimer interactions during clamp closure and also holds the ATP-binding site. The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, respon...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp

Oestergaard

Giangiacomo²,

Bjergbæk³

et al. 2004

“…Plasmid pBY105 contains the yeast TPI promoter inserted into the polylinker region of the LEU2/ARS-CEN plasmid pRS315, which was used as the backbone for pHT212 and pHT⌬350 -407, carrying the wild-type human TOP2␣ cDNA and the human TOP2␣ cDNA with a deletion spanning amino acids 350 -407, respectively. Both pHT212 and pHT⌬350 -407 contain a bicomposite tag at the C-terminal end consisting of the c-Myc epitope and a hexahistidine tail (9). Modified versions of YEpWOB6 were used for overexpression of the hexahistidine-tagged human topoisomerase II␣ and h⌬350 -407 enzymes.…”

Section: Methodsmentioning

confidence: 99%

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

Bjergbæk¹,

Kingma²,

Nielsen³

et al. 2000

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...

“…Topo IIA 3 enzymes share similar core domains and the corresponding catalytic "two-gate" mechanism: passing one segment of duplex DNA (the transfer or T-segment) through a transient double-stranded break in a second segment of DNA (the gate or G-segment) in an ATP-dependent process (5, 6, 8 -10). Catalytically dispensable C-terminal domains (CTDs) are structurally diverse among Topo IIA enzymes, but are important for cellular function as they contain phosphorylation sites and nuclear localization signals (11)(12)(13)(14)(15)(16). Growing evidence suggests that CTD variations among Topo IIA enzymes also alter the topological specificity and activity of the otherwise similar core domain functions of prokaryotic and eukaryotic Topo IIA enzymes (17)(18)(19)(20)(21)(22)(23).…”

mentioning

confidence: 99%

Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

Seol

Gentry

Osheroff

et al. 2013

Background: Human topoisomerase II␣ unlinks catenated chromosomes and preferentially relaxes positive supercoils. Results: Supercoil chirality, twist density, and tension determine topoisomerase II␣ relaxation rate and processivity. Conclusion: Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains. Significance: Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase II␣.