Characterization of new DNA topoisomerase 1 mutants able to modulate the camptothecin anticancer drug reactivity

Human topoisomerase 1B (hTop1) controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes, such as DNA replication, transcription, recombination and repair. HTop1, which is composed of four major domains (N-terminal, core, linker and Cterminal domains), catalyzes the relaxation of negative and positive DNA supercoils, by introducing a transient single-strand break and a covalent link with the 3’-phosphotyrosil end of the broken DNA strand. Top1 is a significant medical interest since it is the only target of anticancer drugs, such as camptothecin and its water-soluble derivatives that specifically and reversibly bind to the transient covalent enzyme–DNA complex inhibiting the rotation and religation steps of the catalysis. Stalled Top1 is a threat to genomic stability as it can be converted into DNA strands breaks and irreversible damage can occur upon collision with the replication fork (Pommier, 2013). In this thesis we have characterized some new hTop1 mutants able to modulate the enzyme reactivity toward the anticancer drug camptothecin. In detail, the functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to CPT as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural–dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and drug resistance are two correlated events. The role of the Gly717 residue, located in an α-helix bridging the active site and the linker domain, has been investigated mutating it into Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of CPT toward the mutant. The role of Gly717 has been investigated inserting in its place a negatively charged amino acid, i.e. an aspartic acid. The mutation gives rise to a hypersensitive CPT mutant when expressed in a DNA repair deficient yeast strain. In vitro religation kinetics assay shows that the mutant is characterized by a fast religation rate, that it is quite significantly reduced in the presence of CPT. Molecular dynamics simulation studies indicate that the Gly717Asp mutant modifies the architecture of the CPT binding site along the trajectory, increasing its affinity for the drug. This result explains the high CPT persistence observed in the Gly717Asp cleavage complex as determined through an equilibrium experiment.