Control of Strand Scission by Type IIA Topoisomerases

Topoisomerases are a family of essential enzymes that disentangle chromosomes and manage DNA supercoils, and are targets of a broad class of successful antibiotics and anticancer therapeutics. The type IIA topoisomerases operate by a complex ATP-dependent strand passage mechanism in which the enzyme transports one DNA segment through a transient, enzyme-mediated break in a second DNA segment. To understand the mechanism of DNA cleavage by type IIA topoisomerases, I used a suicide DNA substrate to crystallize the DNA binding and cleavage core of the enzyme covalently bound to DNA through its active-site catalytic tyrosine. The crystal structure revealed that type II and IA topoisomerases employ a novel variation of canonical two-metal ion phosphoryl-transfer chemistry to achieve DNA cleavage. Additionally, the suicide DNA substrate enabled me to determine the first structure of a fully-catalytic type IIA topo II-DNA-nucleotide complex. The structure revealed the overall doubly-domain swapped architecture of the enzyme. This organization produces an unexpected interaction between the bound DNA and a conformational transducing element in the ATPase domain, which is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. The data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset. Lastly, the DNA-bound structure of one of the two human topo II isoforms, topo IIα, underscores the coupling between the DNA cleavage active site configuration with both metal ion occupancy at the cleavage center, as well as the dimerization status of a key dissociable interface over 50 Å away. The structure also highlights amino acid differences in the drug-binding pocket between the two human isoforms that could serve as differentiating features for developing more selective anti-topoisomerase agents. The work presented in this dissertation helps to explain years of biochemical studies, unifies many elements of topo II mechanism and its control by allostery, and has implications for both understanding large ATP-dependent DNA-remodeling molecular machines as a whole, as well as understanding the means by which small molecules target these enzymes for clinical benefit