Structural and Biochemical Basis of Etoposide-Resistant Mutations in Topoisomerase IIα

Abstract

Etoposide is a widely used anticancer drug that targets type II topoisomerases, including topoisomerase IIα (TOP2A). TOP2A is a nuclear enzyme involved in regulating DNA topology through a double-strand passage mechanism. TOP2A is a homodimeric enzyme with two symmetrical active sites formed by residues from either half of the dimer. Both active sites cleave DNA, forming an enzyme-bound, double-stranded DNA break. Etoposide acts by binding in the active site between the ends of cleaved DNA, preventing the enzyme from ligating the DNA. In the present study, biochemical and structural data are used to examine the mechanism of etoposide resistance found with specific point mutations in TOP2A. Mutations near the active site (D463A, G534R, R487K), along with some outside of the active site (ΔA429 and P716L), are examined. We hypothesize that changes in the coordination of DNA cleavage results from mutations that impact symmetrical relationships in the active site and surrounding regions. In some cases, we report the first data on purified versions of these enzymes. Based upon our results, both local and long-distance factors can impact etoposide action and may indicate interdependent relationships in structure and function.