The tail that wags the dog: topoisomerase IV ParC C-terminal domain controls strand passage activity through multipartite topology-dependent interactions with DNA.

Type IIA Topoisomerases are essential enzymes that contribute to chromosomal integrity by controlling the degree of supercoiling and segregating newly replicated chromosomes1. They share a conserved core mechanism in which a double-stranded segment of DNA is passed through a transient double-stranded break in a second segment of DNA (Figure 1). Remarkably, this conserved strand passage reaction results in a range of distinct activities catalyzed by different but related type IIA topoisomerases2. At one extreme is prokaryotic DNA gyrase that negatively supercoils DNA, but is a poor decatenase. At the other extreme is the closely related prokaryotic topoisomerase IV that is an efficient decatenase and relaxes positive supercoils more efficiently than negative supercoils1. Eukaryotic type IIA topoisomerases display similar, though less extreme, topologically-dependent differences in activity3. For example, human topoisomerase IIα preferentially relaxes positively supercoiled DNA, whereas the other human isoform, topoisomerase IIβ, relaxes positively and negatively supercoiled DNA with equal efficiency. The differences in activity among these enzymes can largely be attributed to differences in the poorly conserved C-terminal domains (CTDs), which contain a highly positively charged DNA binding surface4. Deletion of the gyrase GyrA CTD or topoisomerase IV ParC CTD results in a type IIA topoisomerase lacking chiral preference in relaxation and which no longer introduces negative supercoils4; 5. Indeed, chiral discrimination by topoisomerase IIA enzymes appears to be entirely dictated by the CTD domain. The chirality-dependent activity of chimeras between the human topoisomerase II α and β core enzymes with the alternative CTDs is almost entirely dictated by the CTDs rather than the core enzyme6. In addition, subtle mutations in a gyrase GyrA CTD result in an enzyme that exhibits topoisomerase IV-like chirality-dependent strand passage activity7. Whereas the domain responsible for chiral discrimination has been identified, the mechanistic basis for chiral discrimination by type IIA topoisomerases remains under debate. Proposed mechanisms include discrimination based on DNA crossing geometry and chirality-dependent differences in processivity4; 8; 9; 10; 11. Crystal structures of bacterial GyrA CTDs and ParC CTDs have not provided a great deal of additional insight. The GyrA and ParC CTDs are multi-bladed structures that contain a highly positively charged region that binds DNA 12. The similarity of the five blades found in E. coli ParC CTD suggests that it may simply provide an extended high-affinity DNA binding surface4; 12. Thus, despite determining the structure of the domain responsible for chiral discrimination by topoisomerase IV, the relationship between the structure and mechanism was not immediately clear. Figure 1 Topoisomerase IIA strand passage reaction. Topoisomerase binds gate segment DNA (blue) followed by binding of transfer segment DNA (yellow) and ATP that closes the N-gate (light blue). The gate segment is cleaved and the C-gate (light green) opens allowing ... In this issue, Vos, Lee, and Berger present the results of a systematic dissection of the role of key residues in each of the five blades of the topoisomerase IV Par-C CTD. By measuring the chirality-dependent relaxation and unlinking activity of point mutations of highly conserved basic residues in each blade, complemented by binding and bending assays of the intact enzyme as well as the isolated CTD, the authors made a number of striking discoveries. Rather than being a monolithic DNA binding element, each blade in the CTD appears to affect different aspects of topoisomerase IV activity in dramatically different manners. These results provide an important connection between the structure of the CTD and chirality-dependent modulation of strand passage activity. Remarkably, interactions between the DNA and the Par-C CTD appear to both stimulate and inhibit specific topology and substrate-dependent activities of topoisomerase IV. Perhaps the most interesting finding is that residues in blade 1, which is proximal to the N-terminal portion of the ParC domain, contribute to bending of the G-segment DNA bound by the core enzyme. Disruption of this residue leads to a severe decrease in activity across all measurements, consistent with the growing body of evidence establishing the importance of DNA bending for topoisomerase IIA activity13; 14. However, this is the first evidence that the ParC CTD participates in G-segment DNA interactions and it explains the dramatic decrease in activity observed in the topoisomerase IV ParC CTD deletion mutant4. The remaining blades modulate the activity of topoisomerase IV in distinct DNA substrate-dependent manners. Conserved basic residues in blades 2–4 modulate the activity, rate, and processivity of positive supercoil relaxation. Mutations of these residues decrease the overall activity of topoisomerase IV in relaxing positive supercoils. Paradoxically, these mutations slightly increase the relaxation rate while decreasing the processivity of positive supercoil relaxation. Mutations in blade 5 have little overall effect on the relaxation of positive supercoils. Remarkably, blade 5 appears to specifically inhibit negative supercoil relaxation as both the overall activity and rate increase when interactions between this blade and the DNA are disrupted. Negative supercoil relaxation appears to be governed by blades 2 and 3 as mutations in these blades decrease the overall activity, processivity, and rate of topoisomerase IV in relaxing negative supercoils. Mutations in blade 4 have virtually no effect on negative supercoil relaxation. Similar to negative supercoil relaxation, decatenation appears to be inhibited through the interaction of DNA with blade 5, but only blade 3 appears to be important for decatenation. Mutations in blades 2 and 4 have essentially no effect on decatenation activity or rate. The final surprising discovery by Vos and co-workers is that the effects of disrupting DNA binding at each blade did not uniformly decrease the overall binding affinity of the isolated CTD. Blades 1 and 2 appear to have little effect on DNA binding, whereas blades 3, 4, and 5 appear to play important roles in DNA binding, with mutations in blade 4 showing the strongest effect.