Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB

Abstract

Topoisomerases relieve the torsional strain in DNA that is built up during replication and transcription. They are vital for cell proliferation and are a target for poisoning by anti-cancer drugs. Type IB topoisomerase (TopIB) forms a protein clamp around the DNA duplex and creates a transient nick that permits removal of supercoils. Using real-time single-molecule observation, we show that TopIB releases supercoils by a swivel mechanism that involves friction between the rotating DNA and the enzyme cavity: that is, the DNA does not freely rotate. Unlike a nicking enzyme, TopIB does not release all the supercoils at once, but it typically does so in multiple steps. The number of supercoils removed per step follows an exponential distribution. The enzyme is found to be torque-sensitive, as the mean number of supercoils per step increases with the torque stored in the DNA. We propose a model for topoisomerization in which the torque drives the DNA rotation over a rugged periodic energy landscape in which the topoisomerase has a small but quantifiable probability to religate the DNA once per turn.

The dynamic interplay between DNA topoisomerases and DNA topology

“…Topo V (topoisomerase Type IC) shares a similar catalytic mechanism with Topo IB despite the lack of structural or sequence similarity (Schoeffler and Berger 2008). DNA torsion lowers the rotational energy barrier (Wereszczynski and Andricioaei 2010) and thus enhances the rate of rotation, i.e., the relaxation rate for both types of topoisomerases (Koster et al 2005;Seol et al 2012;Taneja et al 2007). DNA twist also affects the efficacy of human Topo IB inhibitors, some of which are Federal Drug Administration-approved chemotherapeutic agents that act by preventing religation of the cleaved DNA and trapping the cleavage complex comprised of the topoisomerase, DNA, and inhibitor (Pommier and Cushman 2009).…”

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements

“…Since their invention (28)(29)(30), MT techniques have often been used to analyze the interactions between DNA and DNA ligands, including small drug molecules (25,31,32) and proteins (33)(34)(35)(36)(37)(38). MT can be used to apply controlled force and torsion to single DNA molecules and enables measurement of the influence of such nanomechanical stresses on the DNA structure, primarily through the assessment of end-to-end distance at nanometric resolution.…”

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

“…In the rejoining reaction, the 5 0 -hydroxyl group acts as a nucleophile to attack the 3 0 -phosphotyrosyl linkage and restores the continuity of the double helix (Stewart et al, 1998;Krogh and Shuman, 2000). As this 'strand rotation' mechanism of type-1B topoisomerases functions without energetic cofactor, only DNA torque and friction drive one or several integral rotations of the duplex (Koster et al, 2005). The type-2 topoisomerases, that is, the eukaryotic topo II (encoded by TOP2), are functional homodimers that use ATP to transport one DNA duplex through a transient doublestrand break in another duplex (Wang, 1998).…”

“…Yet, a deeper analysis of their normal contributions is impaired by the complexity of in vivo experimental systems and the multiple functions in which they are involved. So far, the catalytic efficiency of eukaryotic topoisomerases has been examined in vitro on naked DNA molecules, by using supercoiled plasmids and DNA single-molecule assemblies (Charvin et al, 2003;Koster et al, 2005). However, their activities on nucleosomal DNA, their natural substrate, have not been contrasted.…”