STUDIES ON MECHANISM OF REPAIR OF ULTRAVIOLET-IRRADIATED VIRAL AND BACTERIAL DNA IN VIVO AND IN VITRO

Abstract

The formation of thymine dimers [Formula: see text] from adjacent intrastrand thymines by ultraviolet (UV) irradiation in DNA was studied under different conditions. When thymine-2-C14DNA was exposed in quartz tubes to 0.5 × 106 ergs/mm2ultraviolet irradiation, two photoproducts were formed: "a" + [Formula: see text]. The concentration formed in dry DNA was only about [Formula: see text] of that formed in wet DNA.The survival of T1phage in the dark in the resistant (uvr+) and the sensitive (uvr−) mutants of E. coli K-12 after UV irradiation of the phage in the wet and dry state is markedly dependent on the state of the phage during irradiation. Survival of T1phage, when UV irradiated dry, was the same in the sensitive as in the resistant host cells, over a wide range of UV doses, while a marked difference in sensitivity existed when it was UV irradiated wet. Similar survival was obtained also by photoreactivation. These results correspond with the notion that thymine dimers are involved both in photoreactivation and in dark (host cell) reactivation.Thymine-requiring E. coli K-12 cells were mutated to a radioresistant strain uvr+(AB 2416) and a radiosensitive strain uvr−(AB 2419).Irradiation of cells with a dose of 1000 ergs/mm2, followed by incubation of the cells in the dark in enriched M9 media, stopped by addition of 5% trichloroacetic acid, hydrolysis of the acid-insoluble fraction and the acid-soluble fraction in trifluoroacetic acid at 175 °C, and separation of the products by paper chromatography, showed that the two irradiation products "a" + [Formula: see text], which were formed in the bacterial DNA, are excised from the DNA in the uvr+strain and appear in the acid-soluble fraction. No such excision occurred upon incubation of the radiosensitive strain uvr−.Incubation of the cells under light showed that photoreactivation prevails in the radiosensitive strain, i.e. disappearance of "a" + [Formula: see text] from the DNA, without their appearance in the acid-soluble fraction, while dark reactivation prevailed in the uvr+strain, a result that indicates a stronger affinity of the dark reactivating system to UV-irradiated DNA. Cell extracts prepared by breaking the cells in a French press in Tris buffer, pH 7.5, plus 10−3 M Mg++plus 10−3 M mercapto-ethanol showed a similar mechanism; the two irradiation products were excised from DNA by a uvr+cell extract and not by a uvr−cell extract. Extract of uvr+cells brought about excision of photoproducts from DNA of the uvr−cell extract.The results suggest that an enzyme, capable of excising thymine dimers, is present in the radioresistant cell as part of the system of repair of DNA from UV irradiation, and its mechanism is demonstrated, both in vivo and in vitro.