Highly purified S. aureus DNA gyrase is a type II topoisomerase encoded by two genes GyrA and GyrB from staphylococcus aureus. DNA gyrase is an essential topoisomerase that supercoils DNA through a process of strand breakage/resealing and DNA wrapping. As a type II enzyme, gyrase is unique in its ability to negatively supercoil a relaxed plasmid DNA substrate. S. aureus DNA gyrase is also the target for quinolone-based antibacterial agents which act by subverting the enzyme into a DNA damaging agent. TopoGEN offers purified DNA gyrase from the gram positive bacterium, Staphylococcus aureus for use in all aspects of drug development and screening assays. The enzyme is very active in decatenating and supercoiling kinetoplast DNA (kDNA) but will also negatively supercoil relaxed plasmid DNA. The S. aureus enzyme is a heterotetramer of GyrA2GyrB2 and is purified as His tagged subunits that are reassembled to make active enzyme.
DNA gyrase subunits were cloned overexpressed and purified using the proprietary company methods. A single band on SDS-PAGE was detected by CB staining for each subunit. Cross contamination by topo I was assessed by assaying for relaxation of supercoiled DNA under conditions optimized for type I activity. Under these conditions, after 2 hours of incubation with supercoiled plasmid DNA, no relaxation products were detectable.
SA gyrase is shipped on dry ice and should be stored at -70°C. It is stable undiluted for at least 6 months in this concentrated state. The enzyme can be aliquoted on first thawing to minimize damage from multiple freeze thaw cycles.
SA DNA Gyrase Reviews and Citations
Phillips JW, Goetz MA, Smith SK, Zink DL, Polishook J, Onishi R, Salowe S, Wiltsie J, Allocco J, Sigmund J, Dorso K, Lee S, Skwish S, de la Cruz M, Martin J, Vicente F, Genilloud O, Lu J, Painter RE, Young K, Overbye K, Donald RGK, Singh SB: Discovery of Kibdelomycin, A Potent New Class of Bacterial Type II Topoisomerase Inhibitor by Chemical-Genetic Profiling in Staphylococcus aureus. Cell Chemistry and Biology 2011, 18: 955-965. doi: 10.1016/j.chembiol.2011.06.011.
Peng H, Marians K: DNA Topoisomerase Protocols Volume I 1999. p. 163 TGVi
Wang J: DNA Topoisomerases. Ann. Rev. Biochem 1996, 65:635-692