Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: II. In vivo studies with Escherichia coli cells and bacteriophage

Abstract

Intracellular complexes of UV lesions in DNA with molecules of photoreactivating enzyme (PRE) in E. coli can be photolysed with a probability close to 1 by single light flashes of about 1 millisecond duration. The observed photoreactivation (PR) effect permits the number of PRE-substrate complexes present at the time of the flash to be determined on the following basis: ( a) The number of PRE-substrate complexes equals the number of lesions repaired; ( b) The number of photorepairable UV lesions present in DNA equals the number of pyrimidine dimers recoverable from acid hydrolysates; a UV dose of 1 erg·mm −2 of 2537 Å radiation causes the formation of approximately 6.5 dimers per E. coli chromosome; ( c) The observed PR effect can be expressed by the formal “dose-reduction”, i.e. by the reduction in the number of dimers. The kinetics of intracellular complex formation can be followed by varying the time interval between UV irradiation and flash reactivation. Stationary phase B s−1 cells, irradiated with 4.8 erg·mm −2 form a maximum number of complexes within approximately 5 min at room temperature, 50% of them being formed within the first 10–15 sec. For greater UV doses (6.4–24 erg·mm −2) the maximum number formed reaches a constant limiting value of 20, indicating that this is approximately the number of PRE molecules in these cells. Experiments with sequential flashes at various temperatures between 2° and 44° show that both the maximum extent of complex formation and the photolytic rate constant are temperature-independent in this range. Hence, the usually observed temperature-dependence of PR under conditions of continuous illumination reflects the temperature-dependence of the complex formation only. Extensive PR effects with single light flashes were also found in UV-irradiated phage T1 after injection of unirradiated B s−1 cells. The effects are much smaller in irradiated host cells due to competition by the bacterial DNA. Creating the competitive substrate after the phage DNA has reacted with the PRE results in a time-dependent decrease of the PR effect in phage as the enzyme equilibrates between the host-cell and phage DNA's. The slow rate of equilibration indicates a relatively high stability of the complexes in the dark. A heterogeneity of the photoproducts is evident with regard to both complex formation and stability. Comparative experiments with phage infecting other host strains indicate that the number of PRE molecules in strain B/r equals that in B s−1, but is lower in the K12 derivatives AB 1157, AB 2437 and AB 2480.