E. coli DNA Gyrase Drug Screening Kit Background
This kit contains purified bacterial (E. coli) DNA Gyrase purified to homogeneity (>98% based on SDS-PAGE). The Gyrase is prepared from overexpressing strains and is supplied as purified holoenzyme in an A2B2 complex. The enzyme is supplied at the unit concentration given on the quality control data provided with the kit. It is also stored in a stabilization buffer [50 mM Tris-Cl pH 7.5, 100 mM KCl, 2 mM dithiotreitol, 1 mM EDTA, 50% glycerol]. Included is substrate relaxed DNA (plasmid pHOT1). This kit is designed to screen for novel gyrase targeting drugs, either as interfacial poisons (or IFPs that induce cleavage of DNA) Catalytic inhibitory compounds (CICs that block gyrase action by any number of mechanisms). It is a straight forward gel assay kit that is easily adopted.
The DNA Supercoiling Assay: This drug screening Kit is based on gyrase supercoiling action on a relaxed plasmid DNA substrate provided (pHOT1, which is a derivative of pBR322) and roughly 2.7 KB. One unit of gyrase will supercoil 200-500 ng in 1 hr at 37oC under conditions defined below. The reaction mechanism is describe below in Fig. 1. The enzyme introduces negative supercoils into a relaxed pHOT1 plasmid substrate via a sign inversion model. An energy co-factor (ATP) is required for the complete supercoiling reaction. The inset gel image in Fig. 1 represents a typical substrate and product (supercoiled DNA) result based on an agarose gel that lacks ethidium bromide. These non-EB gels are ideal for detecting gyrase activity as attested by the excellent resolution between supercoiled and relaxed DNA forms.
Drug Screening Assay. This kit is designed to identify IFP and CIC (poisons and inhibitors, respectively) using the assay described in Fig. 1. A typical IFP (e.g., a fluoroquinolone) will induce double strand DNA breaks while a prototypical CIC will simply block the enyzme from acting on relaxed DNA (novobiocin, some intercalators for example). Thus, an IFP (ciprofloxacin) will result in formation of a linear DNA product while a CIC will block supercoiling activity. These events can easily be detected with this kit.
Kit Contents (100 Assays):
1. Relaxed pHOT1 DNA (50 ug total) DNA concentration is specified on page 1.
2. Supercoiled pHOT1 DNA (25 ul in gel loading buffer). Load 2 ul as a marker.
3. Linearized pHOT1 DNA (25 ul in gel loading buffer). Load 2 ul as a marker for cleavage (DS DNA breaks).
4. 5x Gyrase assay buffer (600 ul) 1x buffer contains recipe is given above.
5. Dilution buffer (600 ul). Dilute gyrase with this buffer. Recipe for dilution buffer given above.
6. 10x gel loading buffer (300 ul): 0.25% bromophenol blue, 50% glycerol, use 0.1 volume in reactions.
7. 10x Proteinase K (100 ul) at 0.5 mg/ml (10x stock of proteinase K and should be diluted to a final 1x to degrade proteins after reaction termination; i.e. use at 50 ug/mL final).
8. 10% SDS (300 ul). To be added to reaction mix to give a final concentration of 1% to terminate the relaxation reactions.
9. Purified DNA gyrase; 100 units (unit definition given on page 1).
10. Ciprofoxacin 10ug/ml: 50 ul (10x Stock; dilute 1:10 in final reaction mix to give 1 ug/ml final).
DNA Gyrase Quality Control. QC testing is performed on our purified enzyme:
1. A test for nuclease contamination was carried out by assaying for the formation of linear Kinetoplast DNA (kDNA) and linear plasmid DNA. Incubations of 1 µg of catenated kDNA or supercoiled pUC19 DNA (4 hrs. at 37°C in the presence of 10 mM MgCl2) were performed. Linear DNA or breakdown products were not generated under these conditions.
2. The A and B subunits are >98% pure based upon SDS-PAGE and certified to be endonuclease free.
3. Note: with high input levels of DNA gyrase, a small amount of linear DNA may be detected due to spontaneously aborted reactions. This is normal and actually serves to work as an internal marker for linear DNA.
The kit may be shipped at ambient temperature or on ice (dry ice or wet ice). The DNAs should be stored at 4°C and the buffers stored at -20°C upon receipt. Avoid frequent freeze/thaw cycles with the plasmid as this may contribute to DNA breakage.