Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells.
We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.
Thomas
D C
DC
Laboratory of Molecular Pharmacology, National Cancer Institute, Bethesda, Maryland 20892.
Okumoto
D S
DS
Sancar
A
A
Bohr
V A
VA
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000528:radiation effects*; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D005260:Female; D005796:Genes; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D010053:Ovary; D011740:Pyrimidine Dimers / Q000302:isolation & purification; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase