6632 Nucleic Acids Research, 2005, Vol. 33, No. 20 However, this stretching barrier is small compared to the over- all protein breathing motions (10–14 A˚ ), and therefore relaxa- tion of negative supercoils should not be inhibited. A protein that performs the reverse function, allowing relaxation of pos- itive supercoils and preventing that of negative ones, may be obtained by engineering disulﬁde bonds in the hinge region. A possible pair of Cys mutants could involve, for example, Leu429 and Lys436, which have their Cas only 6 A˚ away from each other in our equilibrium simulations. This hinge- clamping strategy is likely to allow relaxation of positive supercoils without much impediment, because the hinge region is unchanged during the relaxation of positive super- coils. A ﬂuorescence resonance energy transfer (FRET) experi- ment, with two ﬂuorophores, one in the upper and one in the lower cap, could constitute another test of the proposed dis- tinct conformational changes (72). To observe opening, the ﬂuorophore pair would be placed next to the lips or next to the hinge. A technical issue to be resolved would be to ﬁnd that pair of ﬂuorophore labels for which the Fo¨ rster distance of the donor-acceptor pair (at which FRET efﬁciency is 50%) would be around 20 A˚ to accommodate an extended hinge or the opening of the lips (see Figure 2). FRET experiments could also possibly shed light in the following matter. The state of the protein upon a full DNA rotation [in both (+) and ( ) cases] is not the same as the state of the protein at 0 . We have no rigorous proof to exclude the formal possibility that this is a consequence of the rapid time scale for the simulation (on which the protein might not have enough time to relax). Such a proof would require extending the simulation to times that are not available to current computer hardware. However, we believe that it is more likely that what we see at 360 is an intermediate state between open and closed. This intermediate would be the state in which rotation of the nicked DNA is allowed. It is important to note in this regard that DNA is thought to rotate several times before religation, as shown by recent single molecule (24) and bulk experiments (68). A transition from this metastable intermediate to the closed state would be fol- lowed by religation. Because such a transition can well take beyond microseconds, we were not able to observe it by direct molecular dynamics. It is also imaginable that another inter- mediate exists that would allow sliding along the DNA duplex, in-between two relaxation events. The conformations we have generated at 360 could also be representative of such a putat- ive state (although if it exists, it should correspond to a non- covalent complex). Another way to alter activity by altering ﬂexibility and dynamics can be attempted in a systematic way by changing amino acids throughout the protein, gauging their ﬂexibility by normal modes, and performing more detailed molecular dynamics studies on those particular mutants that show a signiﬁcantly altered pattern of projections onto rotation coordinates. This is particularly important because recent evidence points to coupled networks of predominantly con- served residues that inﬂuence protein motion, which in turn points at an evolutionary selection of these coupled networks, of potential importance to protein engineering (67). Topo I may be used as a biological machine that selectively relaxes only one type of supercoil, while preventing the other at speciﬁc-sites on the DNA chain. In an instance of a topo IB enzyme with speciﬁcity to the DNA sequence to which it binds, vaccinia topoisomerases, a possible ‘strangling machine’ could in principle be devised to alter preferentially the sign of supercoiling in a sequence-dependent fashion and possibly switch genes on and off. Human topoisomerase I is the sole target of a novel class of potent anti-cancer drugs from the CPT family. Recent evid- ence (73) points to the fact that CPT, in addition to impeding religation, might also hinder rotation by intercalating at the nick site. Additional studies of the type presented here applied to the CPT-bound system should be important in understand- ing in detail the role of this anti-cancer drug. SUPPLEMENTARY DATA Supplementary Data are available at NAR online. ACKNOWLEDGEMENTS This work was supported from funds from the University of Michigan. Computational resources from the National Science Foundation (TeraGrid) are kindly acknowledged. We thank Professor J. J. 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