Topoisomerase IB Activity Investigated By Single Molecule Magnetic Tweezers: Mechanisms of Cytotoxicity

Magnetic Tweezers (MT) are a powerful tool to investigate how single topoisomerase IB (topIB) molecules relax DNA supercoils [1,2]. MT studies have revealed that topIB activity is dramatically affected by the presence of camptothecin-class (CPT) inhibitors, used clinically as anti-cancer drugs [3]. In the presence of CPT, topIB remains covalently bound to DNA for much longer than in the absence of the drug (>100 s vs. ∼ 2 s) and the rate of supercoil removal is significantly reduced, in particular for positive supercoils. The CPT-induced asymmetry in the rate of supercoil removal between positive and negative supercoils leads to an accumulation of positive supercoils in the G1 and S-phases in yeast cells in vivo [3].Here, we present results on the G365C topIB point mutant, which exhibits CPT resistance and shows no accumulation of positive supercoils in vivo. In the MT assay in the absence of CPT, the G365C mutant shows activity similar to wt topIB. In the presence of CPT, G365C exhibits long-lived DNA-topIB complexes and slow supercoil removal for positive supercoils, similar to the wt enzyme. Surprisingly, for negative supercoils we found similarly long-lived complexes and slow supercoil removal for the G365C mutant. In contrast to the wt enzyme, the G365C mutant removes positive and negative supercoils with similar (slow) velocities in the presence of CPT. These results suggest that CPT cytotoxicity might be more strongly dependent on the asymmetry of the rate of positive vs. negative positive supercoil removal and the corresponding accumulation of positive supercoils than on the lifetime of the covalent DNA-topIB complex.[1] Koster, et al. Nature 2005[2] Lipfert, et al. Meth. Mol. Biol. 2009[3] Koster, et al. Nature 2007