The interaction of gyrase with DNA: Biochemical and structural characterization

DNA gyrase is an essential bacterial protein that acts in concert with other topoisomerases to modulate DNA topology. Nearly all processes that act on DNA rely on this indispensable modulation, including replication and transcription. Each of the bacterial topoisomerases has a distinct cellar role. Gyrase is unique in its ability to increase the topological stress of DNA and does so unidirectionally, introducing only negative supercoils. It was previously known that this activity was dependant on DNA wrapping by the holoenzyme and that this somehow involved the C-terminal domain of GyrA.; Upon structural characterization by our lab and others of the C-terminal domain in GyrA and the homologous ParC, a model where the GyrA-CTD directly wraps DNA for presentation to the holoenzyme core began to emerge. Differences in the structures from different organisms, however, alluded to variation in gyrase mechanism. We compared the GvrA-CTDs from E. coli and B. burgdorferi and determined that the topology by which they wrap DNA differs correspondingly to the path of hypothetical DNA binding for each structure. This allowed us to propose a more precise model where, in E. coli, the GvrA-CTD binds DNA with right-handed ((+) solenoidal) superhelicity that enforces presentation of a (+) node to the cleavage-reunion core, leading to the introduction of (-) supercoils after T-segment passage via nodal sign inversion. In B. burgdorferi, placement of the GyrA-CTDs in relation to the rest of the holoenzyme is responsible for orientating bound DNA so that only (+) nodes are presented.; We further characterized binding of DNA by the GyrA-CTD using a number of orthogonal methods including fluorescence resonance energy transfer, single-molecule lambda-DNA shortening microscopy, electrophoretic gel shift assays, and surface plasmon resonance. The most surprising result was that the equilibrium dissociation constant for binding of a 45-mer with the GyrA-CTD was 4.0 nM, while full GyrA binding of DNA (35 to 170-mers) alone was so weak that it could not readily be measured by gelshift. We hypothesized that the highly-negatively charged C-terminus of GyrA could competitively inhibit DNA binding by GyrA in the absence of GyrB. GyrA deletion mutants with residues removed from the C-terminus confirmed this, in that they have increasing affinity for DNA that corresponds to the deletion length. Ultimately, the terminal 34 amino acids of GyrA, (841-875), were shown to inhibit DNA binding by the rest of GyrA and furthermore, the GyrA-CTD. This stretch of amino acids can also inhibit binding intermolecularly, albeit weakly. Furthermore, the inhibition is sequence dependant. The pSC101 strong gyrase site can overcome the inhibition and bind GyrA with an equilibrium dissociation constant of 16 nM.; In designing the original construct for GyrA-CTD crystallization, most of the C-terminal inhibitory amino acids had been removed, as they were predicted to have no secondary-structure. Protein from a new GyrA-CTD construct extending to the most C-terminal amino acid was crystallized in hope that the structural basis for C-terminal inhibition of DNA binding could be determined. When solved, however, most of the C-terminal amino acids were disordered. Large differences between the two structures were located at packing interfaces, showing that yet another packing arrangement yields the same unique spiraling quaternary structure. The presence of a previously unseen large packing interface (2245 A2 of buried surface area) gives a structural basis for the higher ordered configurations of GyrA-CTD and DNA we observed in gel shifts and lambda-DNA shortening.; To the end of obtaining structures of gyrase with DNA, targeted disulfide trapping of DNA with GyrA was completed. This strategy has yet to yield highly diffracting crystals.