6628 Nucleic Acids Research, 2005, Vol. 33, No. 20 Figure 3. (A) Interaction energies (locally-averaged, and in kcal/mol) of DNA with the linker domain (residues 636–712) as a function of the extent of rotation. (B) Interaction energy of DNA with the right nose-cone helix (a5, Thr303 to Gln318). (C) Interaction energy of DNA with left nose-cone helix (a6, Lys321 to Tyr338). (D) The variation of the distance (in angstroms) of the distal clamp (His367–Ala499) in all four systems, as a function of the extent of rotation of downstream DNA. (E) The corresponding plot for the proximal clamp (Gly365–Ser534). All distances are measured between the two Ca atoms. Legends in inset are the same for all panels. ﬁnal changes can be regarded as negligible when compared to those obtained in rotations for removal of positive supercoils where the changes are observed around 10–14 A˚ (see Figure 3D and E). Given that (i) our simulations indicate that the behavior of the protein acting on negative supercoils is fundamentally distinct from the positive case, and that (ii) clamping experiments were performed on positive super- coils, it would be of great future interest to perform the two lip-clamping experiments with negatively supercoiled DNA substrates. Possible implications of the N-terminal domain Deletion of the ﬁrst 190 amino acids from the N-terminus of human topo I (amino acids 1–190) is proved not to modify signiﬁcantly human topo I’s mechanism relative to the full length enzyme (47–49). However, three recent studies showed results that bring about remarkable insights when viewed from the point of the simulations reported here. Firstly, in 2001, Lisby et al. (50), by truncating amino acids from the N- terminal all the way down to position 206, proved that the