The ATPase Reaction Cycle of Yeast DNA Topoisomerase II: SLOW RATES OF ATP RESYNTHESIS AND PiRELEASE

Cheryl L Baird,Matthew S Gordon,David M Andrenyak,James F Marecek,Janet E Lindsley

doi:10.1074/jbc.m102544200

Cheryl L Baird, Matthew S Gordon + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m102544200

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Abstract

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism. Two key rates in the ATPase mechanism, ATP resynthesis and phosphate release, were investigated using 18O exchange and stopped-flow phosphate release experiments, respectively. The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release. When topoisomerase II was bound to DNA, phosphate was released slowly, with a lag. Since each of the preceding steps is known to occur rapidly, phosphate release is apparently a rate-determining step. The length of the lag phase was unaffected by etoposide, indicating that inhibiting DNA religation inhibits the ATPase reaction cycle at some step following phosphate release. By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as approximately 0.5 s(-1). These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II.

Highlights

DNA topoisomerase II catalyzes the transport of one DNA duplex through a transient break in a second duplex using a complex ATP hydrolysis mechanism
The 18O exchange results showed that the rate of ATP resynthesis on the topoisomerase II active site was slow compared with the rate of phosphate release
The rate of DNA transport was determined from rapid quench decatenation experiments and is consistent with transport occurring after hydrolysis of one ATP and before hydrolysis of the second [9]

Summary

SLOW RATES OF ATP RESYNTHESIS AND Pi RELEASE*

By combining the 18O exchange and phosphate release results, the rate constant for ATP resynthesis can be calculated as ϳ0.5 s؊1 These data support the mechanism of sequential hydrolysis of two ATP by DNA topoisomerase II. By contrast, when the reactions are chemically quenched, as opposed to being chased with unlabeled ATP, the burst amplitude equals half of the enzyme active site concentration [8] This was the first evidence that topoisomerase II binds two ATPs, but only hydrolyzes one rapidly, before a rate-determining step in the mechanism. These results indicate that yeast topoisomerase II binds two ATPs, hydrolyzes one, transports DNA, and releases the products of the first hydrolysis, and, hydrolyzes the remaining ATP Two aspects of this complex ATPase mechanism that remain unknown are the rates of ATP resynthesis and Pi release. These results help to complete our understanding of how DNA topoisomerase II utilizes ATP to transport DNA segments

ATP Resynthesis and Pi Release by Topoisomerase II

RESULTS AND DISCUSSION

Rate constanta