Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VIII. Inhibition of photoenzymatic repair of UV lesions in E. coli DNA by caffeine

Abstract

The rate of photoenzymatic repair (PER) obtained with continuous illumination of UV-irradiated E. coli B s−1 cells is much decreased in the presence of 16 mg/ml caffeine. This inhibitory effect is usually not observed in B/r cells. It occurs in T1 phage infecting either B s−1 or B/r, indicating that its absence in B/r cells has not a trivial explanation such as destruction or decreased uptake of the inhibitor. Experimental evidence is presented that caffeine interferes strongly with the formation of enzyme-substrate (ES) complexes, presumably by binding to DNA at, or close to, the repairable photoproducts. This results in a lower equilibrium of complex formation in the dark. A single caffeine molecule can block the formation of a complex; this effect is fully reversible when the caffeine is diluted out. Caffeine does not interfere with the photolysis of existing ES complexes. Failure to observe, under usual conditions, caffeine inhibition of PER in irradiated B/r cells is explained by the large number of UV lesions in these cells, compared to much smaller numbers in B s−1 cells or in phage infecting either type of cell. Due to this high number, complex formation in B/r cells is so rapid that—in spite of the slowdown by caffeine—most of the PRE is tied up in ES complexes in the steady state during continuous illumination at moderate intensity. Only at very high light intensity, where the steady-state concentration of complexes is lower, is the caffeine inhibition observed. Correspondingly, illumination of irradiated B s−1 cells at very low light intensity results in decreased caffeine inhibition of PER.