Excision of uracil from bromodeoxyuridine-substituted and U.V.-irradiated DNA in cultured mouse lymphoma cells

A uracil-DNA glycosylase activity was detected in cell-free extracts from cultured mouse lymphoma L5178Y cells. A study has been made of the role played by this enzyme in the removal of uracil from chromosomal DNA. U.V. light (254nm) irradiation of the cells with BUdR-substituted DNA produced not only single-strand breaks but also 'internal' residues that were recognized as substrate sites by uracil-DNA glycosylase. These 'internal' uracil residues were lost from the DNA upon reincubation of the irradiated cells. The product released from the DNA was identified as uracil. Thus, the intracellular action of the uracil-DNA glycosylase was demonstrated and the subsequent reconstitution of the DNA strand was inferred in cultured mammalian cells. (author)