Repair of individual DNA strands in the hamster dihydrofolate reductase gene after treatment with ultraviolet light, alkylating agents, and cisplatin.

Abstract

We have analyzed gene-specific and strand-specific DNA damage and repair in the dihydrofolate reductase gene in hamster cells. Cells were UV-irradiated or treated with two types of chemotherapeutics, alkylating agents or cisplatin. UV-induced pyrimidine dimers were detected using a previously published technique in which the T4 endonuclease V enzyme is used to create nicks at the lesion sites. 6-4 photoproducts were detected in a similar assay using ABC excinuclease after prior reversal of the pyrimidine dimers with photolyase. Adducts formed by the alkylating agents nitrogen mustard and dimethyl sulfate were quantitated by generating strand breaks at basic sites after neutral depurination. Cisplatin-induced intrastrand adducts were detected with ABC excinuclease, and cisplatin interstrand cross-links were detected using a denaturation-reannealing reaction before electrophoresis. In accord with previous reports by other investigators, we find distinct strand specificity of the repair of pyrimidine dimers after UV; the transcribed strand was much more efficiently repaired than the nontranscribed strand. In contrast, there was little or no strand bias in the repair of the 6-4 photoproducts. For alkylating agents, a slight bias toward repair in the transcribed strand was found after treatment with nitrogen mustard, but there appeared to be no bias in the repair after treatment with dimethyl sulfate. Cisplatin interstrand cross-links are repaired with equal efficiency from the two strands, but the more common cisplatin-induced lesion, the intrastrand adduct, is preferentially repaired from the transcribed strand. In conclusion, there is strand bias in the repair of pyrimidine dimers and cisplatin intrastrand adducts, but the strand specificity of repair may not be a general feature for all DNA lesions, as we found little or no strand bias in the repair of other lesions studied.