STRUCTURAL STUDIES OF TYPE II TOPOISOMERASE FRAGMENTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We plan to collect native data sets on two topoisomerase II fragments. Type II topoisomerases modulate DNA supercoiling and resolve topological crises intrinsic to virtually every phase of DNA metabolism. It is thought that a DNA duplex is passed through a transient enzyme-bridged double stranded break in another DNA segment in order achieve the divers topological changes. As such, topoisomerases represent an Achilles heel of eukaryotic and prokaryotic cells. Indeed, human topoisomerase II inhibitors serve as widely used chemotherapies. The mechanistic basis of this inhibition by small molecules is well understood phenomenologically, yet there is a paucity of structure of any of these drugs in complex with their eukarotic topoisomerase targets. Towards an improved understanding of these interactions and the normal ATP hydrolysis coupled to the mechanical process of DNA duplex transport, we have crystallized the ATPase domain of the alpha isoform of human topoisomerase II. Home source diffraction limits are around 3 A. The recently published yeast topo II ATPase structure should serve as an excellent search model for molecular replacement. DNA gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. This cellular activity is critical to prokaryotic viability, forming the basis of potent and clinically efficacious antibiotic gyrase inhibitors, e.g., ciprofloxacin (Cipro). The negative supercoil induction bias unique to gyrase has been traced to the C-terminal domain of gyrase A. There are tantalizing hints in the literature that this domain actually wraps DNA around it in a putative structure reminiscent of the nucleosome. Indeed the initial structure (our early data collected at A-1 last year) of a mutant and subsequent biochemistry and biophysical measurements have confirmed this finding. We would like to get the fragment structure in the absence of mutations and now have co-crystals with DNA bound (40 bp duplex) before we publish these findings. These structures can explain the negative supercoil induction preference for this important class of proteins.