Structural and functional studies on human DNA Topoisomerase IB: interation with supercoiled DNA and the antitumor drug Camptothecin

DNA Topoisomerases are essentials enzymes in biological process involving the relaxation of the superhelical tension of the DNA molecules. Human DNA topoisomerase I (hTop1p) is a monomeric conserved enzyme of 91 KDa, subdivided into four domains: the N-terminal; the core constituted by three subdomains; the C-terminal domain containing the catalytic residue Tyr723; the linker connecting the core with the C-terminal domain, locating the active site in the catalytic pocket, and characterized by two protruding coiled coil α-helices. DNA relaxation occured with a mechanism of DNA strand rotation in a multiple step process where the 5’-OH end is free to rotate around the intact strand. Two conformational changes are involved in this dynamic process: the enzyme binding the DNA through an open shape, then closed clamp that completely embraces the DNA. No external energy source occurred, but arises from the release of the superhelical tension. In addition hTop1p is of great interest since is the cellular target of the antitumor drug camptothecin (CPT). The reversible binding of the drug to the covalent complex DNA-Top1p brings to lethality due to religation inhibition and double strand breaks formation in a S-phase dependent manner. Different data supported the “controll rotation mechanism” to explain how this enzyme can relax both positevely and negatevely supercoiled substrates. First of all crystal structures [17] of human Top1 with a 22 bp oligonucleotide reported no sufficient space inside the clamp for a free rotation. Moreover a mutant isoform of the protein with 2 cysteines produced opposetely to the lips domains, locked the clamp through a disulfide bridge and reported that an opening conformation was not required for the relaxation activity [21] [135]. Also single-molecule experiments suggested that friction and torque are necessary for the relaxation process and the number of supercoils removed per cleavage-religation cycle is strictly dependent from the number of supercoils of the DNA substrate. The aim of this project is focused on the contribute of key residues located al the level of the hinge and involved in the control rotation mechanism during DNA relaxation. In my experiments Saccharomyces cerevisiae strains were used as a model organism to investigate a series of topoisomerase IB mutants: Pro431Gly; Arg434Ala; Arg434Cys; Trp205Cys. These substitutions are interesting because of their position in the enzyme three-dimensional structure. The flexible hinge (429-436 residues) facilitating clamp opening by a stretching or bending motion pivoting around Pro-431 and the cluster of large aromatic residues around the top of the hinge, probably are involved in controlling the movements of the α-helix and the proper closing of the clamp. In 2005 Sari and Andricioaei simulation analyses reported the possible role of the lips for the relaxation of the positive supercoils and the strechting of the hinge for the negative supercoils removal. Yeast strains lacking for endogenous topoisomerase I gene were transformed with YCpGAL1-hTOP1, YCpGAL1-hTOP1Pro431Gly, YCpGAL1-hTOP1Arg434Cys, YCpGAL1-hTOP1Arg434Ala, YCpGAL1-hTOP1Trp205Cys plasmids and viability was verified in a drug yeast sensitivity assay. Transformants were spotted in presence and absence of camptothecin at different concentrations. Moreover CPT sensitivity for each mutant was analyzed through measures of the stability of the covalent complex formation. Results are congruents and are also supported by Frohlich and Knudsen data, in 2007, suggesting a role for the Trp-205 and surrounding residues in the control of strand rotation during negative but not positive supercoiled removal, and reported a more CPT sensitivity during relaxation of positively versus negatevely supercoils. EKY3 strains expressing htop1W205Cp showed a partial resistance phenotype, and top1R434A, top1R434C mutants are not sensitive to lower drug dose (0.01<ug/ml