aphidicolin and dimethyl-sulfate

Alkylating agents have been reported to give rise to both short and long patches of repair. The reason for the different patch sizes is not known. One possibility is that alkylating agents can trigger both base and nucleotide excision repair. Another possibility is that base excision repair itself can result in different patch sizes. Recognition and incision at lesions is the rate limiting step in excision repair. In order to discriminate between base and nucleotide excision repair it would be desirable to be able to distinguish between different incision activities. In order to accurately measure incision rates, the rejoining of the strand-breaks formed must be inhibited. We have used two inhibitors, aphidicolin and 3-aminobenzamide. Aphidicolin, an inhibitor of DNA polymerases alpha/delta/epsilon. caused accumulation of single-strand breaks both after UV and dimethylsulphate. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose)-polymerase caused accumulation of single-strand breaks only after alkylating agents and is thus specific for base excision repair. Enzymatic activities can be characterised by their activation energy. In order to discriminate between base and nucleotide excision repair the temperature dependence of incision activities was determined. When the temperature is decreased, the incision rate is reduced to a larger extent for UV than for DMS-induced repair. Incisions in UV-irradiated cells are practically cut off at temperatures of 15 degrees C and below, whereas DMS-exposed cells still are actively repairing at this temperature. In DMS treated cells the temperature dependence was the same whether aphidicolin or 3-aminobenzamide was used, speaking against an involvement of nucleotide excision repair. In addition, cell lines deficient in nucleotide excision repair responded in the same way to aphidicolin after DMS treatment as normal cells and were able to make incisions at 15 degrees C. This indicates that nucleotide excision repair is not to any significant amount involved in repair of DNA damage induced by a methylating agent.

Topics: Aphidicolin; Benzamides; Cells, Cultured; DNA; DNA Damage; DNA Repair; Endodeoxyribonucleases; Enzyme Inhibitors; Eukaryotic Cells; Humans; Multienzyme Complexes; N-Glycosyl Hydrolases; Nucleotides; Purines; Pyrimidines; Sulfuric Acid Esters; Temperature; Ultraviolet Rays

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.

Topics: Aphidicolin; Bleomycin; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; HeLa Cells; Humans; Hydroxyurea; Kinetics; Sulfuric Acid Esters; Thymidine; Ultraviolet Rays; Zinostatin

Topics: Animals; Aphidicolin; Benzamides; Diterpenes; DNA; DNA Repair; Humans; Leukemia L1210; Lymphocytes; Mice; Niacinamide; Sarcoma 180; Sulfuric Acid Esters; Ultraviolet Rays