UC Berkeley

Topoisomerases are essential molecular machines that manage physical challenges presented by DNA topology. Life has evolved two classes of enzyme, type IIA and type IIB topoisomerases, which unlink entangled chromosomes by coordinating a complex, ATP-dependent passage of one DNA segment through a transient double strand break generated in a second DNA segment. Both classes share functional modules and an overall mechanism, yet they possess distinct architectures. Type IIA topoisomerases must coordinate transient strand scission with the association and dissociation of three distinct dimer interfaces, whereas type IIB topoisomerases must accomplish the same using only two interfaces. The biophysical basis of communication between these interfaces underlies the action of frontline small-molecule therapeutics which corrupt these concerted movements. To address how type IIA topoisomerases coordinate strand scission with the dimerization status of their unique third interface, I used X-ray crystallography to determine the first structure of the DNA cleavage core of human topoisomerase II. Comparative analyses to previously determined structures revealed that the principal site of DNA engagement in type IIA topoisomerases undergoes highly-quantized conformational transitions between distinct binding, cleavage, and drug-inhibited states that correlate with the control of subunit-subunit interactions. Additional consideration of this model in light of an etoposide-inhibited complex of human topoisomerase IIβ suggests possible strategies for developing inhibitors specific to topoisomerase II, the primary target of these therapies. To address how type IIB topoisomerases faithfully coordinate strand scission using only two interfaces, I performed biochemical analyses which show that these enzymes actively recognize and utilizes DNA bends and crossings formed in supercoiled DNA to stimulate ATP binding and hydrolysis as a prerequisite for efficient DNA cleavage and strand passage. This finding indicates that binding of two DNA segments preferentially drives the ATP-triggered dimerization, a coordinated action with important ramifications for how both type II topoisomerase machineries control of double-strand break formation.