‘further-down’ residues 191–206 participate in topoisomeriza- tion either by binding DNA directly or by promoting DNA- binding to other regions of the enzyme. The truncated enzyme distinguished itself from the wild-type by exhibiting insens- itivity to the anti-cancer drug CPT in relaxation and inability to ligate blunt end DNA fragments. This implicates the truncated region in the control of strand rotation. Secondly, in 2003, Christensen et al. (51) also reported that residues 190–210 of human topoisomerase I are required for enzyme activity in vivo (but not in vitro). Thirdly, while our work was in progress, Frøhlich et al. (52) strengthened the conclusion found by Lisby et al. (50) by studying the N-terminal residues more in detail, and hypothesized that Trp205 (among other amino acids positioned between 191–206) coordinates DNA rotation during topoisomerization. From a structural point of view, Redinbo et al. (27) had resolved, in 2000, 12 additional residues at the N-terminal domain (positions 203–214). The observations that (i) these new residues were found to be in close proximity of the hinge region, and that (ii) our simulations indicate that relaxation of negative supercoils induces a stretch in the hinge (rather than causing the lips to open) can be corroborated to provide a possible explanation for the biochemical deletion studies presented above. That is, taken together, our ﬁndings and the new N-terminus structural data thus suggest that the N-terminal region is, in fact, important for the removal of negative supercoils because some of its residues are packed against the hinge region. [This region would therefore be a hot spot for engineering mutations either in the hinge or N-linker to alter topo I activity.] More interestingly, Trp205 [found to be particularly important by Frøhlich et al. (52), see above] is the closest residue, among the 12 newly resolved residues, to the hinge region (Leu429 to Lys436): it has side chain atoms only around 4 A˚ away from those of Ser432 and Arg434. We cannot, at the moment, say more about the function of the N-terminal as it is absent from the covalent structure our studies were performed on. Import- antly, however, all three biochemical studies (50–52) on the role of the N-terminal domain were performed on negatively supercoiled DNA. In the light of the different negative versus positive relaxation protein motions seen in our simulations, it is tempting to speculate that the N-terminal region can, in fact, control removal of only negative supercoils (through its close interactions with the hinge). Mutations in the N-terminus should not signiﬁcantly affect positive supercoiling relaxation. [Although there could be ‘second-order’ effects due to the fact the opening motion of the lips hinges on the hinge region. Therefore, for an Cys-mutant experimental test of our predic- tion (i.e. that the hinge stretches upon relaxation of negative turns), it would be optimal if the disulﬁde bond is ‘locked’ after non-covalent binding of the protein (by pH change).] This is a testable hypothesis in future experiments that would compare hinge-mutated topo I activity on positively and negatively supercoiled DNA substrates. RMS deviations during MD simulations In terms of the average root-mean-square deviations observed in all heavy protein atoms, system 1 produced the largest deformations in the linker part, while the core subdomain II shows the minimum RMS deviations, as seen in Figure 4A. Nucleic Acids Research, 2005, Vol. 33, No. 20 6629 This is consistent with the argument that the linker domain is in more control of the relaxation of positive supercoils than the right nose-cone helix (which is within the core subdomain II). This is because the DNA duplex is a double-stranded stiff chain, with persistence length of about 1000 A˚ , so even if we impose the rotation around the intact strand, the protein is capable of adapting to take the orientation of ‘minimal resistance’ during DNA rotation. Therefore, correlation between RMSD and the interaction energies with the DNA suggest that those domains that control the rotations are more ﬂexible, and they adopt the necessary structural changes to conserve the interactions with the rotating DNA atoms. [Sev- eral equilibrium MD simulations on topoisomerases have been recently performed (53–55) and have explored additional cor- relations between domains; however, there was no attempt to model the actual relaxation mechanism (as was done here).] Similarly, both system 2 and 4 (which relax negative super- coils) exhibit a distinct RMSD in core subdomain I (see Figure 4B and D), which is larger than those observed for the other domains. This is also in agreement with the obser- vation that the left nose-cone helix (which is within the core subdomain I) controls the rotation for relaxation of negative supercoils, more than the linker does. The study of Redinbo et al. (26) on protein ﬂexibility concluded that the linker domain and the upper cap region (composed of subdomain I and II) show the maximal degree of ﬂexibility, a result in good agreement with the above discussion. They observed that, at least in the crystal structures, the linker domain is the most ﬂexible part of the protein exhibiting up to 4.6 A˚ non- isomorphic shifts, followed by the cap region with up to 3.6 A˚ deviations. These experimental ﬁndings are consistent with our calculated RMS deviations of the room-temperature solvated system, which are less than 4.5 A˚ , with the largest RMS deviations in the linker and the core-subdomain I (on the upper cap), followed by the C-terminal and core- subdomain III. While we ﬁnd that the RMS deviation of individual domains from their crystal structure reference stays below 4.5 A˚ (i.e. we do not artiﬁcially unfold domains by the rapid rotation), large, see-saw-like opening motions of the two caps as a whole due to the strand rotation are observed. Although large, these dis- placements are comparable to those reported in experiments on other topoisomerases. For Escherichia coli topo I, large conformational shifts of about 20 A˚ are also reported (22). Similarly, type IIA topoisomerases have been shown to under- go dramatic conformational changes that are larger than 20 A˚ (56,57). This paints a rather dynamic picture of the larger family of topoisomerase enzymes in general as they manip- ulate DNA by ample motions. Same set of topoisomerase normal modes encodes both opening motions in a see-saw fashion To gauge the inherent ﬂexibility and large amplitude (low frequency) motions related to the conformational changes that the protein undergoes, and to complement the model provided by the molecular dynamics simulations, we have performed a normal mode analysis of human topoisomerase I on the MD-equilibrated structures. (This consists of the diag- onalization of the second-derivative matrix of the potential energy, yielding the vibrational frequencies, as well as the