Nucleic Acids Research, 2005, Vol. 33, No. 20 6623 Figure 1. (A) DNA-human topoisomerase I covalent complex, seen from linker side of protein and along DNA helical axis. DNA downstream of the nick (see text) in yellow, upstream DNA in purple, protein is in green, (nose-cone helices pointed at by blue lines); lips region, including the 35 residue difference between the distal and proximal clamps (see text and Figure 1D), are in red, hinge region is in orange. (B) Different perspective of DNA-human topoisomerase complex focusing on the DNA duplex. Protein shown in transparent colors to make DNA visible. Active site residue (Tyr723) and the phosphate group at the nick point are represented by ball and stick model to show covalent attachment. The phosphate groups of 1 base (Ade) and and the +10 base (Ade), defining the rotation axis (which is in blue) are shown as large spheres, yellow for the +10 Ade and purple for the 1 Ade. The remaining coloring scheme is the same as in part A. In our molecular dynamics simulations, we rotate downstream DNA duplex (in yellow) around the blue rotation axis [mimicking the rotation driven by the supercoiling torque(24)] as indicated by the double-headed black circular arrow. (C) Caricature of supercoil relaxation, as revealed by simulations. View perspective as in Figure 1A; DNA: yellow cylinder, protein: two green hemispheres (upper cap and bottom part) connected by an orange thread-like hinge (see panels A, B of this figure and text); lips are on the right, at opposite side of the hinge. To bind DNA, the enzyme opens upper and lower ‘jaws’ and closes them around DNA. Following arrows to the left or right describes relaxation of negative or positive DNA supercoils, respectively. To relax negative supercoils, DNA needs to rotate clockwise in the enzyme’s grip; torque applied in our MD simulation in that direction causes stretching of the (orange) hinge region. Counter-clockwise DNA rotation relaxes positive supercoils and produces opening of the lips. (D) The two clamps used in Carey et al. and Woo et al. Downstream DNA: yellow; upstream DNA: purple. The proximal clamp (Woo et al.) was produced by Cys mutations in Gly365 and Ser534, and shown in blue. The distal clamp (Carey et al.) was engineered by mutations in His367 and Ala499, and is colored green. The remaining lips of the protein is in red.