and the linker (in the lower cap) are thought (21) to control DNA rotation within the protein because these domains are positively charged. The two nose-cone helices sit oriented along the spiral groove of the rotating DNA duplex and, depending on the direction of rotation, impart a push to different domains of the surrounding protein, much like the way a nut around a screw is pushed in different parts of its inner lining when the screw rotates clock or anti- clockwise. The DNA rotation that relieves positive supercoils affects mainly the right nose-cone helix a5 (i.e. on the right side of Figure 2A, see also movie 1), which pushes open the right side of the cap region. On the other hand, the rotation that relaxes negative supercoils moves ﬁrst the left nose-cone helix a6 (in Figure 2B, see also movie 2), inducing motion of the left cap region, which in turn stretches the hinge. Interaction energies along rotation coordinate The energy of interaction of DNA with the right nose-cone helix, left nose-cone helix, as well as with the linker domain, is calculated along the rotations. In agreement with the above observation, they show that the linker domain and the right nose-cone control the rotation of the DNA pre- dominantly for removal of positive supercoils, whereas the left nose-cone helix (a6) controls rotations that relax negative supercoils. This prompts at residue locations for charge-neutralization mutations with the potential to alter relaxation of one type of supercoiling, but not of the other. As seen in Figure 3A, the interaction energy (95% of which was electrostatic) between the linker and DNA is conserved during rotations that relax positive supercoils (system 1 and system 3 in Table 1), whereas this interaction is lost during rotations that relax negative supercoils (systems 2 and 4 in Table 1). However, the situation is reversed for the interaction between DNA and the left nose-cone helix (see Figure 3C), in which the interaction quickly weakens for positive supercoil relaxation (system 1 and system 3), while it is conserved for negative relaxation (system 2 and system 4). [Due to ﬁnite sampling problems (common to all molecular dynamics simu- lations of large systems), the actual values of the energies presented here are to be taken in a qualitative sense. A more detailed energy analysis is to be done in future work using potential of mean force calculations.] For the interaction between DNA with the right nose-cone helix, the two simultaneous 180 rotations of both parts (sys- tem 3, see Table 1) produced conservation of the interaction as expected. However, a 360 rotation of just the downstream part (system 1 in Table 1) shows no such conservation (see Figure 3B). This can be a result of the fact that the interaction between the linker domain and the DNA is more stable in system 1 than in system 3, meaning that the linker is in control of positive supercoil relaxation. Again, the right nose-cone helix-DNA interaction is almost completely lost in systems 2 and 4 (see Figure 3B), showing that the left nose-cone helix, and not the right one, dominantly controls rotations that relax negative supercoils. The above ﬁnding is in agreement with the recent single molecule experiment on vaccinia topo I (24), which revealed that friction between protein and DNA is important in the Nucleic Acids Research, 2005, Vol. 33, No. 20 6627 control of DNA rotation. As friction derives, on the atomic scale, from the interaction energy between the topoisomerase cavity and DNA, our aforementioned discussion on the pre- servation of the interaction supports the conclusion of the friction-based model proposed by Koster et al. (24). In addi- tion to it, we have revealed speciﬁc sites in the protein that govern friction (e.g. the linker and nose-cone helices), and we surmise that, without the protein, DNA rotation might be faster, but certainly ‘uncontrolled’. Analysis of clamping studies The fact that the disulphide clamping studies of Carey et al. (45) and Woo et al. (46) produced opposite results (the proximal clamp inhibited relaxation, while the distal one did not) was interpreted to arise from the relative positioning of the clamps with respect to the rotating DNA. Although Carey et al. used an N-terminal truncated human topoi- somerase I (topo70), while Woo et al. used a full length pro- tein, the truncated topo70 (starting from residue 175) retained its activity. The fact that the ﬁrst study used temperature changes and the second a DNA intercalator is not likely to be fundamentally signiﬁcant, as both of the procedures have the same effect on the DNA substrate. The explanation of the observed differences is most likely that the proximal clamp strangles DNA more tightly than the distal one, imped- ing strand rotation. However, no time-resolved details of the entire rotation mechanism existed. Moreover, although both studies used only positively supercoiled DNA substrates, [For DNA intercalators, this requirement was due to the imposs- ibility to assay negative supercoils because such studies induce DNA supercoiling after the formation of topo-DNA catenanes. A temperature shift method should, in principle, not have this problem.] their conclusions were extended to the general relaxation mechanism of both kinds of supercoils. This tacit assumption may have stemmed from the knowledge that human topo I can remove both positive and negative super- coils. However, in the light of the distinct opening motions found in the present rotation simulations, it is essential to differentiate between supercoiling signs. The actual simula- tions on the clamped enzyme systems are in progress, and more detailed explanations for the observed contradictory ﬁndings of Carey et al. (45) and Woo et al. (46) will be available at the atomic level. Meanwhile, we have investigated how the distances between the amino acids of the wild-type corresponding to the distal and proximal clamp Cys mutations change in our different molecular dynamics simulations. As mentioned, during rotations that remove negative supercoils we found, remarkably, that the two lips do not open up, and that both the proximal and distal clamp distances remain almost the same. This feature was reproducible, and both our runs that are adept at relaxing negative supercoils exhibited this feature. In system 2, the two clamp separations ﬂuctuate around 5 ± 0.5 A˚ (see Figure 2B), except that the proximal clamp shows a 1.5 A˚ sudden increase during the last 40 rotations. In system 4, the ﬂuctuations are within ±2.5 A˚ and the same for both clamps. The ﬁnal changes (at the end of 360 rotations) are 0.5 A˚ decrease in the distal clamp and 1.3 A˚ increase in the proximal clamp for system 2, and 0.4 A˚ increase in distal clamp and 0.2 A˚ decrease in proximal clamp in system 4 (see Figure 3D and E). Therefore, these very small