Linus Pauling Institute

To study the effect of ultraviolet irradiation on S and G2 phases of the cell cycle, BB88 mouse cells synchronized by a double thymidine block were exposed to ultraviolet light, and rates of DNA synthesis and mitotic indexes were determined at regular intervals. It was found that with increasing ultraviolet dose, semiconservative DNA synthesis decreased and the sharp mitotic wave observed in the unirradiated cells gradually degenerated. To study repair, semiconservative DNA replication was inhibited with hydroxyurea at different time intervals after releasing cells from the block and the DNA synthesized as a result of repair of the ultraviolet damage was labeled with 5'-bromodeoxyuridine (BrdU). The newly repaired DNA was separated from bulk DNA by immunoprecipitation with monoclonal anti-BrdU antibody, labeled with 32P and hybridized to nine different gene and oncogene probes dot-blotted in excess on nylon membranes to determine their abundance in the repaired DNA. The results showed that: (a) the most actively repaired segment was a 211-bp sequence adjacent to the promotor region of the beta-actin gene; (b) all transcriptionally active genes were repaired at similar and constant rates throughout S and G2 phases; (c) the nontranscribed genes were repaired at much lower rates in early S phase, but later in S phase and especially in G2 phase, their repair rates increased and approached those of the transcribed genes.