Assay of Repair Enzyme Activity by Reactivation of Ultraviolet-Irradiated Infective Viral DNA

Abstract

Treatment of ØX174 replicative form (RF) DNA, pre-exposed to ultraviolet light, with T4 endonuclease V led to a marked increase of infectivity of the RF when the activity was assayed on CaCl₂-treated cells of Escherichia coli strain defective in uvrA gene. The reaction was specific and the extent of the reactivation was proportional to the concentration of the enzyme. Based on this finding, we developed a procedure to assay endonuclease activities specific for ultraviolet-damaged DNA, that might be involved in the incision step of excision repair of pyrimidine dimers. To find conditions suitable for accurate and rapid assays, we examined conditions affecting transfection with ØX174 RF The maximum transfection was achieved when more than 2×10⁸ CaCl²-treated cells, which had been prepared from bacteria harvested during the early or mid-logarithmic phase of growth in L broth, were incubated with the DNA at 0°C for 20 min in 50 mM CaCl₂. Incubation of the cell-DNA mixture at 37°C decreased the transfection efficiency to about 30% of the optimal level; thus, heat shock, a step regarded as necessary in the conventional “CaCl₂ methods” for transfection and transformation, was eliminated. The CaCl₂-treated cells remained viable and competent after storage at -20°C in a solution containing 15% glycerol. By using the procedure thus established, repair endonuclease activities in crude extracts of T4-infected E. coli and of Micrococcus luteus were determined. The procedure should be of use in assaying and purifying repair enzymes of other organisms.