The DNA cleavage/ligation reaction of human topoisomerase IIalpha: New insights into enzyme function and drug mechanism

In order to solve the topological dilemmas generated by the essential processes of replication and transcription, topoisomerase II must cut and ligate DNA in a tightly controlled fashion. The DNA cleavage/ligation reaction of topoisomerase II is also the target of several clinically important anticancer agents. Therefore, the DNA cleavage/ligation cycle of topoisomerase II and the mechanism of action of antineoplastic drugs that poison the enzyme were examined in greater detail.; First, the effects of temperature on topoisomerase II-mediated DNA cleavage indicate that the enzyme undergoes two transitions at high temperature and suggest that temperature may represent a tool for further dissecting the catalytic mechanism of topoisomerase II.; Next, all current assays that examine DNA rejoining by topoisomerase II are limited by the fact that topoisomerase II must cleave the DNA before ligation can be monitored. Thus, a novel cleavage-independent ligation assay was developed to examine the DNA ligation reaction of topoisomerase II in the complete absence of scission. Results demonstrate that topoisomerase II possesses an intrinsic specificity for DNA ligation that parallels its sequence preference for DNA cleavage. Furthermore, this specificity appears to be driven by chemical steps rather than initial DNA binding.; Lastly, the molecular details that underlie the mechanisms of topoisomerase II-targeted drugs are poorly understood. Experiments with the anticancer drug etoposide, which inhibits topoisomerase II-mediated DNA ligation, reveal that this drug must act at both scissile bonds to stabilize a double-stranded DNA break. In contrast, the presence of the quinolone CP-115,953 (which stimulates the forward rate of DNA scission) at one DNA strand is sufficient to stimulate the formation of a double-stranded break. These findings have important implications for cancer chemotherapy, the cellular processing of drug-stabilized topoisomerase II-associated breaks, and the catalytic mechanism of topoisomerase II. Moreover, they underscore the mechanistic differences between drugs that inhibit DNA ligation and enhance DNA cleavage and suggest that there may be important differences in the cellular pathways used to sense and repair the genomic damage generated by these drugs.