How Do Type II Topoisomerases Use ATP Hydrolysis to Simplify DNA Topology beyond Equilibrium? Investigating the Relaxation Reaction of Nonsupercoiling Type II Topoisomerases

Abstract

DNA topoisomerases control the topology of DNA (e.g. the level of supercoiling) in all cells. Type IIA topoisomerases are ATP-dependent enzymes that have been shown to simplify the topology of their DNA substrates to a level beyond that expected at equilibrium (i.e. more relaxed than the product of relaxation by ATP-independent enzymes, such as type I topoisomerases, or a lower than equilibrium level of catenation). The mechanism of this effect is currently unknown, although several models have been suggested. We have analysed the DNA relaxation reactions of type II topoisomerases to further explore this phenomenon. We find that all type IIA topoisomerases tested exhibit the effect to a similar degree and that it is not dependent on the C-terminal domains of the enzymes. As recently reported, the type IIB topoisomerase, topo VI (which is only distantly related to the type IIA enzymes), does not exhibit topology simplification. We find that topology simplification is not significantly dependent on circle size in the range ~2–9 kbp, and is not altered by reducing the free energy available from ATP hydrolysis by varying the ATP:ADP ratio. A direct test of one model (DNA tracking, i.e. sliding of a protein clamp along DNA to trap supercoils) suggests that this is unlikely to be the explanation for the effect. We conclude that geometric selection of DNA segments by the enzymes is likely to be a primary source of the effect but that it is possible that other factors contribute. We also speculate whether topology simplification might simply be an evolutionary relic, with no adaptive significance.

In front of and behind the replication fork: bacterial type IIA topoisomerases

“…In addition, the simplification process appears to be independent of circle size, which also disfavors tracking mechanisms [34,82]. Moreover, the amount of free energy obtainable from ATP hydrolysis does not affect the extent of topological simplification, suggesting an energy facilitated geometric (or kinetic) selection of T segments for unidirectional strand passage [34,82].…”

“…However, simultaneous binding of the enzyme to two juxtapositions is an unlikely event that would dramatically slow down disentanglement [49,75]. In addition, the CTD regions principally responsible for DNA bending do not appear to contribute to DNA structure simplification [82]. Indeed, CTDs would provide an ideal physical location for bent T-segment selection to effectively recognize hooked crossovers.…”

The dynamic interplay between DNA topoisomerases and DNA topology

“…This supercoildependent affinity, in conjunction with other existing models, may shed light on the non-equilibrium topology simplification conundrum. Whereas the below-equilibrium removal of knots and plectonemes may contribute to genomic stability, the temporal dynamics of the process and the exceedingly low energy conversion efficiency (Stuchinskaya et al 2009) suggest that it may reflect some yet to be determined aspect of Type IIA mechanism that may become clear when the fundamental process of below-equilibrium topology simplification is established and subsequently abolished through mutation.…”

“Breaking Up Is Hard to Do”: The Formation and Resolution of Sister Chromatid Intertwines

“…Mechanistically, this has been proposed to occur through bending of the segment of DNA that is broken, thus biasing selection of the DNA segment to be transferred through the break to catenated rather than uncatenated DNA molecules [52]. However, the quantitated shift in equilibrium dynamics is not sufficient to fully decatenate sister chromatids (discussed by Stuchinskaya et al [53]). …”