Type II topoisomerase-DNA interactions: Structure, mechanism, and covalent trapping.

摘要

Type II DNA topoisomerases play a critical role in chromosome supercoil relaxation, decatenation, and condensation by performing the remarkable feat of passing one DNA duplex though another. Although there are a number of fragment structures for these enzymes, none of them contain the DNA---thus our understanding of this acrobatic catalytic cycle and the molecular mechanisms of the many clinically deployed pharmacological agents that target the DNA-protein ternary complex remain limited. While conventional type II topoisomerases act to simplify DNA topology, a prokaryotic member of the topoisomerase II family, DNA gyrase, is actually able to introduce negative supercoils in an ATP dependent manner. Indirectly implicated in this unique ability of gyrase to generate negative supercoils was the C-terminal domain of gyrase A.; I solved the X-ray structure of the E. coli gyrase A C-terminal domain (CTD) with MAD phasing. The spiraling spatial arrangement of blade motifs led naturally to a hypothesis that explains the role of this domain in the context of the full protein. Fragment biochemistry (EMSA determination of dissociation constants and topoisomerase I relaxation assays) and biophysics (single molecule lambda-DNA shortening assays) was used to confirm the hypothesized functional role of this fragment: this domain wraps DNA with a (+) solenoidal writhe and presents the conserved cleavage-reunion core of the protein with a positive node. The strict enforcement of positive node positioning underlies the unique bias for (-) supercoil induction of gyrase. To examine the wrapping in molecular detail with crystallography, a pool-deconvolution disulfide cross-linking strategy was developed.; Type II topoisomerases display only modest sequence specificity---the challenge of obtaining a DNA-bound structure is the generation of homogeneously bound DNA requisite for crystallization. A number of strategies have been employed to generate uniformly trapped species that represent different discrete intermediates in the duplex passage mechanism: disulfide cross-linking with 5'-phosphate tethered thiols, trapping with suicide DNA substrates (nicked species with lesions and bridging 3'-phosphorothioates), and cruciform DNA complexes. Several of these modified DNA oligonucleotides required or allowed innovation of new nucleoside synthetic methodologies. Many of these trapping techniques have been successfully employed to generate various DNA-bound structures with high homogeneity, however, thus far these none of these complexes have produced diffraction quality crystals.