Structure and function of DNA topoisomerase II beta

Abstract

Human topoisomerase II beta (TOP2B) is a type II topoisomerase enzyme that modulates DNA topology using the energy from ATP hydrolysis. During catalysis a transient DNA double strand break is made so that another DNA duplex can be passed through the break relieving torsional strain. TOP2B is a homodimer comprising an Nterminal ATPase domain, a breakage reunion core and a C-terminal domain. The current study investigated the tertiary structure of the ATPase domain of TOP2B and the conformational changes that occur during ATP hydrolysis. The ATPase domain (45-444) was crystallised in the presence of the non-hydrolysable ATP analogue AMPPNP and the ATP hydrolysis product ADP. Moreover, as bisdioxopiperazines are clinically relevant catalytic inhibitors of topoisomerase II that target the ATPase domain, the structure was determined bound to ICRF193, the most potent bisdioxopiperazine. The TOP2B structures reported here lacked the complete N-terminal strap, starting at residue 45. As the strap is the most variable region of the ATPase domain, biochemical characterisation of the full-length ATPase domain (1- 444) and the alternative splice variant (1-449) was performed which revealed the strap is inhibitory to ATP hydrolysis. An increasing number of TOP2B mutations, causative for disease are being identified. Patients can exhibit varying phenotypes from B-cell immunodeficiency to neurological defects like autism. Mutations have been identified throughout the protein, though many concentrate within the TOPRIM domain of the breakage reunion core. Moreover, TOP2B mutations can confer resistance to topoisomerase II targeting drugs. Therefore, to understand the impact of these mutations on enzyme activity, biochemical analysis was performed utilising an in vivo yeast complementation system and in vitro analysis of the purified proteins (45-1621). Generally, conservative residue changes were tolerated although not all patient mutations were functional in vivo. The in vitro catalytic activity was variable with some mutations more detrimental than others, potentially accounting for the different phenotypes observed clinically.