All tangled up: how cells direct, manage and exploit topoisomerase function

Abstract

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.

Fig 1. DNA cleavage and type I topoisomerase mechanisms

(A) Schematic of negative (−) and positive (+) plectonemic DNA supercoiling. The two forms can be distinguished by their right- and left-handed superhelical wrapping, respectively. (B) Schematic of the DNA cleavage reaction. Topoisomerases catalyze strand scission by forming a reversible, covalent enzyme–DNA adduct through their active site tyrosine. Type IB and IC topoisomerases become attached to 3′ DNA ends, and type IA and II topoisomerases attach to 5′ DNA ends. (C) Type IA topoisomerases pass a single-stranded DNA segment (yellow) through a transient break in a second, single DNA strand (green). The structure of E. coli topo III (a type IA topoisomerase) bound to single-stranded DNA is shown . (D) Type IB topoisomerases nick one DNA strand (yellow), allowing one duplex end (green strand) to rotate with respect to the other around the remaining phosphodiester bond. The structure of human topo IB bound to duplex DNA is shown .

Fig 2. Type II topoisomerase mechanism

(A) Type IIA topoisomerases cleave both strands of a duplex DNA (green) and pass another duplex DNA (purple) through the transient break in a reaction coupled to ATP turnover. The cleaved strands are religated, and the products of the reaction are released from the enzyme. The movement of the purple DNA duplex is indicated by a dashed arrow. The DNA cleavage domains are homologous to those of type IA topoisomerases. The structure of the yeast topo II ATPase and DNA cleavage domains are shown ^,. (B) Type IIB topoisomerases use a duplex strand passage mechanism similar to that of type IIA enzymes, and share the same ATPase and cleavage domains but differ in overall tertiary structure. The structure of the topo VI holoenzyme is shown .

Fig 3. Topoisomerase functions during DNA replication

Schematic of topological problems that arise during DNA replication. The names of the topoisomerases that resolve these superstructures are listed. Topoisomerase action is indicated by a dashed arrow. (A) Replication elongation. As a replisome progresses, positive supercoils form ahead of the fork, and newly-replicated precatenanes behind it. If unresolved, precatenanes can give rise to tangled or catenated DNAs that can lead to abnormal DNA segregation upon entry into cell division. Unresolved positive supercoils can impede fork progression and terminate DNA replication prematurely. (B) Replication termination. Hemicatenanes are formed as two forks converge, and these must be resolved before chromosome segregation can occur. The unreplicated mother duplex can be resolved by topo III, together with a RecQ-family helicase (for example, the BLM helicase in eukaryotes), after which the single-stranded gaps are filled in (1). Alternatively, the unreplicated mother duplex can be replicated to form duplex linkages, which are then removed by a type II topoisomerase (2).

Fig 4. Topoisomerase functions during transcription and DNA repair

(A) Schematic of topological problems that arise during transcription. As RNA polymerase progresses, positive and negative supercoils form ahead and behind it, respectively. The names of the topoisomerases that act on these superstructures are listed; topoisomerase action is indicated by a dashed arrow. Topo IV can remove negative supercoils; however, it is listed in brackets because it is not the primary enzyme used by cells to resolve these superstructures. (B) Double-strand break repair through homologous recombination. The repair of broken DNA ends can proceed through a pathway that leads to the formation of a double-Holliday junction (HJ). Topo III, together with a RecQ-type helicase (such as BLM in eukaryotes) can resolve these junctions, generating disentangled chromosomes that have no crossovers between DNA ends (1). By contrast, the resolution of Holliday junctions by branch-endonucleases have a 50% chance of giving rise to repair chromosomes, the arms of which are now swapped (2).

Fig 5. Inhibition or poisoning points for type II topoisomerases

Detailed schematic of the type II topoisomerase reaction cycle, showing all of the points where exogenous agents can disrupt function. During its reaction cycle, the topoisomerase initially binds one duplex DNA segment. Following binding, the topoisomerase can then associate with a second duplex DNA segment. ATP binding stimulates cleavage and opening of the first DNA, and passage of the second through the opening. Agents have been identified that interfere with: (1) DNA binding, (2) ATP binding, (3) DNA cleavage, (4) strand passage, (5) religation, and (6) product release. Only a partial list of all inhibitors and poisons is shown.