Structural insights into the gating of DNA passage by the topoisomerase II DNA-gate

Type IIA topoisomerases (Top2s) manipulate the handedness of DNA crossovers by introducing a transient and protein-linked double-strand break in one DNA duplex, termed the DNA-gate, whose opening allows another DNA segment to be transported through to change the DNA topology. Despite the central importance of this gate-opening event to Top2 function, the DNA-gate in all reported structures of Top2-DNA complexes is in the closed state. Here we present the crystal structure of a human Top2 DNA-gate in an open conformation, which not only reveals structural characteristics of its DNA-conducting path, but also uncovers unexpected yet functionally significant conformational changes associated with gate-opening. This structure further implicates Top2’s preference for a left-handed DNA braid and allows the construction of a model representing the initial entry of another DNA duplex into the DNA-gate. Steered molecular dynamics calculations suggests the Top2-catalyzed DNA passage may be achieved by a rocker-switch-type movement of the DNA-gate.

The MD simulations are technically sound. Pictures are clear. The manuscript is w ell presented; it allows to capture the main message of the story. Reading the manuscript, however, I w as expecting to find something about the presence and role of catalytic metal ion(s). Indeed, MD simulations (including steered MD for drug binding at the cleavage complex) have been used already in the past to look at the dynamics of topoII for function and inhibition (see e. that this open state occurs after DNA cleavage, and therefore corresponds to a more 'flexible" state where ions are likely to be highly movable. However, I invite the authors to comment on this aspect, which I think would enrich the overall story.
One more question regards the model shown at step 3, in figure 5. This is the result of the simulations of the passage of the T-segment, which leads to the structural model that is reported. Maybe it is because of the viewpoint shown in the figure, but this model seems quite distorted compared to the other ones in the same figure. The protein's shape seems somehow squeezed, with the T-segment included at the center. The authors say "the upper portion of the channel continues to narrow, ultimately closing the N-facing entry to the DNA-gate". Is this conformation of this upper portion any similar to the crystallographic one at step 1 ? I w ould appreciate the authors' comment on this particular conformation, and the way it is formed.
The reported structure is of the beta isoform of TopoII. The authors report (abstract and page 9, lines 245-256) that the new structure in part explains w hy topo2 preferentially relaxes positive supercoils. However, as shown by Osheroff and co-workers (Reference 32), the alpha isoform shows a preference for positive supercoils. On the other hand, it seems that the beta isoform (the structure reported in this paper) shows no such preference. Further, Timsit reported (Nucleic Acids Res. 2011 Nov; 39(20): 8665-8676) that for the initial clamping of the G-and T-segments, righthanded crosses are optimal. For the T-segment expulsion, repulsive interactions between the DNA segments were proposed in a left-handed cross-over. Any comments on this?
Minor Corrections: 1. Page 8, Line 212-There is a mention of A'α19 in Fig 3a. How ever, the helix is not marked in the figure. The X-ray crystallography has been very carefully executed with iodo-U substituted DNAs use to unambiguously determine the positioning of the DNA and hence the veracity of the structures The refinement has also been very carefully performed and the structures are well refined.
I could see the crystallographic parameters for the P3sub2 structure in the supplementary, but not that for the P2sub1 structure talked about in the text but the crystallographic parameters are not tabulated. Also in the text the space group is referred to as P3 and not P3sub2! This should be corrected as P3 is also a possible space group (also the Laue group) but the systematic absences and MR provide the definitive space group as being P3sub2.
The steered molecular dynamics simulations are very interesting but I am very surprised that a pair of the helices forming the C-gate completely unwrap at the end of the simulation. I have run molecular dynamics on systems although not on these topo II-DNA and topo II-DNA-drug systems, but to me this w ould be intuitively unlikely as these are very long helices stabilised by many H bonds between the carbonyls and the main chain NHs all along the length of the helices. Are the force field constraints being correctly applied at these ending stages ? Is this a true reflection of what happens in solution, this unwrapping on these time scales ? I think that this paper is a extremely important contribution to the field of topoisomerase structure and mechanism We greatly appreciate the obvious time and effort that the Reviewers spent on our manuscript.
And thanks to their keen attention and valuable comments, we believe our manuscript is further improved after revisions. We thank the reviewer for directing us to these highly relevant and insightful papers, which are now referenced in the revised manuscript (references #49 and #55). where the bound metal ion is coordinated by both Asp residues of the DxD motif. It is widely accepted that the A-site metal ion is essential for G-segment cleavage and religation by stabilizing the penta-coordinate transition state 11,12,22 . In contrast, the functional significance for having a metal ion present in the B-site during catalysis is more controversial; one speculation is that its presence may help anchoring the substrate DNA to enhance cleavage efficiency 12 . Whether Top2 requires simultaneous binding of two metal ions or a single metal ion that shuffles between the two sites has remained unsettled. Nevertheless, the existence of a metal ion in the A-site is observed only when Top2 assumes the pre-cleavage or presumably the immediate post-cleavage state 11,12,22 , in which the scissile phosphate and DxD motif are optimally aligned for metal ion coordination ( Supplementary Fig. 3, panels b and c).
Following G-segment cleavage, the +1 and -1 nucleotides are free to move away from each other. In the structure commonly adopted by the Top2 cleavage complexes 8,11,20,23,28,29 ( Supplementary Fig. 3, panels d and e), the integrity of the A-site is disrupted due to a wider separation between the +1 and -1 nucleotides, in these cases the metal ion is seen exclusively The Reviewer's points are well taken. As the T-segment moves through the DNA-gate and toward the C-gate, the upper portion of DNA-gate becomes narrower, and the lower portion becomes wider. At the same time, the central cavity, enclosed by the helix bundles connecting the DNA-and the C-gate, becomes wider and flatter, creating the "squeezed" appearance in conformation 3. However, the conformation of each half-DNA gate changes little, relative to the crystal structure, as shown by the newly added RMSD plots in Supplementary Figure 8a; thus, we are seeing mainly a rearrangement of the two protomers' relative position and orientation, as now stated in the revised text (Supplementary Figure 8, caption). Furthermore, as mentioned in the original and revised text (shown below), this structure resembles a previously reported crystal structure of Bacillus gyrase, as shown in Supplementary Fig. 8b and cited in reference #40. Thus, we would argue that the conformation in step 3 is plausible.
Page 11, lines 312 ~ 316: "While the quaternary structure of the OC state appears quite different from the ON state, the conformation of each half-DNA gate in the former changes little relative to the latter, as shown by the RMSD plots ( Supplementary Fig. 8a). This indicates we are seeing mainly a rearrangement of the two protomers' relative position and orientation."  2005 351: 545-61), we concluded that the controversy pointed out by the reviewer was resulted from the use of seemingly contrastive terms for describing the handedness of DNA crossings. Specifically, the DNA crossing geometry seen in the (+) supercoiled DNA molecule was referred to as "right-handed crossover" by Timsit (see Fig. 2, NAR 2011 39: 8665-76), but was called "left-handed braid (L-braid)", "left-handed superhelix", or "superhelix with left-handed configuration" by Cozzarelli (see Fig. 1, PNAS 2003 100: 8654-59), Croquette (see Fig. 1, PNAS 2003 100: 9820-25), and Berger (see Fig. 1, JMB 2005 351: 545-61). Since it appears that the term "left-handed" are more commonly used to describe (+) supercoils, we decided to follow this convention in our paper. Nevertheless, to avoid potential confusion regarding this issue, the use of "left-handed DNA crossover" have been replaced by "left-handed DNA braid" in Abstract as well as in text. In addition, we have commented on and put Timsit's proposal in the context of our new structure (shown below).
Page 9, lines 258 ~ 263: "Given that the formation of the L-braid is energetically more favorable than the R-braid 37 , the T-segment may spontaneously dock onto the G-segment with a L-braid-like crossing geometry even before the DNA-conducting channel is formed, and the T-segment can instantaneously enter the channel without the need to reorient as soon as the DNA-gate opens." The reviewer's concern regarding whether the human Top2β exhibits a preference for (+) supercoils is well taken. Our argument is that we thought a small but likely significant preference of Top2β for (+) supercoils can be recognized in the Figure 6 of the Osheroff's 2005 JBC paper (reference #35; #32 in the original submission). Given that this so-called "chirality-sensing" is mainly mediated by the C-terminal domain that is diverged between Top2α and Top2β, it is reasonable to expect that the preference seen in Top2β would be much smaller, which is why we say the structural feature observed in the new conformation "may" contributes "in part" to the distinguish between (+) and (-) supercoils. We will have no objection to take out this speculation if the reviewer feels it is more appropriate to do so.

Response to Reviewer #2:
[This is a very interesting structural study in the topo II area as it is a first structure

determination of an open G-gate.]
We appreciate this positive assessment of the present contribution. We appreciate this positive assessment on the quality of the reported structures.
[I could see the crystallographic parameters for the P3sub2 structure in the supplementary, but not that for the P2sub1 structure talked about in the text but the crystallographic parameters are not tabulated. Also in the text the space group is referred to as P3 and not P3sub2! This should be corrected as P3 is also a possible space group (also the Laue group) but the systematic absences and MR provide the definitive space group as being P3sub2.] We thank the reviewer for pointing out these problems. Indeed, the P2sub1 structure and a second P3sub221 structure, determined using the 5-iododeoxyuridine-labeled DNA, were used in this study as a part of the effort to verify the formation of Top2 cleavage complex. The atomic coordinates and structure factors of these two structures have been deposited in the Protein Data Bank (PDBid: 5ZQF and 5ZRF). The data collection and refinement statistics for these two structures are reported in the revised Supplementary Table 1. We apologize for not identifying the space group properly in the initial submission. The correct space group should be P3sub221. We have fixed this problem in the revised manuscript. The Reviewer's points are well taken. Our simulations were carried out using force field parameters described in a prior study (Nucleic Acids Res 2015, 43(14): 6772-6786). An unbiased microsecond molecular dynamics simulation was presented in that work, and these alpha-helices remained well folded. In the steered simulations presented in the current study, we applied positional and distance restraints to accelerate the strand passage process, which probably occurs on a considerably longer time-scale in nature. We did not apply additional constraints to the C-gate helices, because we aimed to see how the protein's conformation would respond to complete strand passage with minimal added perturbations. We are not aware of experimental evidence for or against the unwinding of these helices during the catalytic cycle. However, there is precedent for local helix unwinding in a DNA-binding enzyme, as the central bridge helix of RNA polymerase I (Pol I) can adopt either a fully folded conformation, in the elongation complex, or an unwound state, in the free enzyme states, one may notice that the coiled-coil region underwent a helix-to-coil transition in the latter. We are not aware of experimental evidence for or against the unwinding of these helices during the catalytic cycle of Top2. However, there are precedents for functionally relevant local helix unwinding events. For example, the central bridge helix of RNA polymerase I can adopt either a fully folded conformation, in the elongation complex, or an