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

Abstract

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...

Dissecting the Cell-killing Mechanism of the Topoisomerase II-targeting Drug ICRF-193

“…In contrast to 408i, the wild-type enzyme as well as 351i and Y805S caught the circular plasmid DNA in the presence of ATP and ICRF-193 (Fig. 3), in accordance with the previously reported clamp-closing capabilities of the enzymes (11,19,20,23). Thus, the inability of ICRF-193 to cause cytotoxicity in vivo in yeast cells expressing 351i and Y805S does not correlate with an inability of ICRF-193 to stabilize the closed clamp complex in 351i and Y805S.…”

“…These observations demonstrated that the cytotoxicity of m-AMSA is independent of topoisomerase II activity and solely results from the formation of topoisomerase II-DNA cleavage complexes. Furthermore, our data revealed that 351i and 408i, besides being expressed, are capable of mediating DNA cleavage in the yeast cells, in agreement with their in vitro abilities (19,20). DISCUSSION Recently, several studies have focused on the topoisomerase II-directed ICRF compounds, and accumulating evidence that these drugs work as a type of topoisomerase II poison has been presented (10 -15).…”

“…The enzymes are 351i (19), 408i (20), and the active site mutant Y805S (2). 351i has a two-amino acid insertion at position 351 in the transducer domain.…”

“…In this study, we have used three human topoisomerase II␣ mutant enzymes to determine the catalytic steps/activities of human topoisomerase II␣ through which ICRF and m-AMSA exert cell killing: (i) a mutant enzyme unable to perform DNA strand passage but capable of both clamp closure and DNA cleavage (19); (ii) a mutant enzyme unable to close the clamp and perform strand passage but still capable of DNA cleavage (20); and (iii) a topoisomerase II enzyme defective in DNA cleavage and strand passage but able to close the N-terminal clamp (2).…”

Human Topoisomerase IIα Rapidly Relaxes Positively Supercoiled DNA

“…Effects of the Geometry of DNA Supercoils on ATP Hydrolysis by Human Topoisomerase II␣-DNA-bound topoisomerase II hydrolyzes more ATP than does free enzyme (58,(77)(78)(79)(80)(81). It has been proposed that the stimulation of ATP hydrolysis by DNA represents a nonproductive cycling of the enzyme in the "closed clamp" form and actually decreases the efficiency of ATP utilization (81).…”