Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation.

Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in 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 camptothecin toward the mutant.
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