Abstract
Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.
Highlights
Introduction of theG164I Point Mutation—Two rounds of PCR were carried out to introduce the G164I point mutation in the human topoisomerase II␣ cDNA
The current mechanistic model of topoisomerase II catalysis involves a complex sequential mechanism of ATP hydrolysis, where binding and hydrolysis of ATP drive the enzyme through a series of conformations
Whereas hydrolysis of one ATP is thought to accelerate transport of the T-segment through a gate formed upon cleavage of the G-segment, it is presently unclear how the second ATP is correlated to the remaining steps of the catalytic cycle, a connection to the resetting of the enzyme has been suggested [11, 12]
Summary
Introduction of theG164I Point Mutation—Two rounds of PCR were carried out to introduce the G164I point mutation in the human topoisomerase II␣ cDNA. This demonstrates that human topoisomerase II␣ can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP.
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