Microrheology assays to study gelation of circular DNA by TOP2 clamp formation

Abstract

Topoisomerase II (TOP2) regulates DNA entanglement in replication, transcription, and repair. Driven by the hydrolysis of ATP, TOP2 has the ability to grab two strands of DNA and transpose their positions via a cleavage mediated passage reaction. TOP2 is a target for cancer therapeutics, because it is involved in late stages of cell division. In this study, how generic TOP2 inhibitors affect the rheology of a model circular DNA solution was investigated by the microrheology gelation assays. The sol-gel transition of DNA-TOP2 was examined by using multiple particle tracking. By the time-cure superposition the gel kinetics of DNA and TOP2 was analyzed very accurately. Inhibition of TOP2 was examined by the model inhibitor AMP-PNP and the anticancer drug ICRF-193. By using AMP-PNP rather than ATP, a closed clamp was formed by inhibiting the double strand passage reaction of TOP2. The result showed that both critical relaxation exponents and gel times decreased with increasing TOP2 concentration. The change in double strand passage reaction was also examined while increasing ICRF-193 concentration under the presence of ATP. When ICRF-193 was bound to the ATP binding site of TOP2 the catalytic cycle was inhibited. Especially, when a sufficient amount of ICRF-193 was present, the gel formation characteristic was surprisingly similar to the case of AMP-PNP. In this method, we have demonstrated that the microrheology method should be a novel assay to study many other anticancer drugs.