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 IIalpha 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 IIalpha 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.
Highlights
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]
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
Insertion of Two Amino Acids at Position 351 in Human Topoisomerase II␣ Abolishes the Strand Passage Activity of the Enzyme—In a previous study performed by Jensen et al [8], it was observed that insertion of two amino acids at position 351 in human topoisomerase II␣ disrupted the ability of the enzyme to complement growth of a yeast strain in which the endogenous topoisomerase II gene was deleted
Summary
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 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.
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