Gene Specific Damage and Repair after Treatment of Cells with UV and Chemotherapeutical Agents
We have previously demonstrated preferential DNA repair of active genes in mammalian cells. The methodology involves the use of a specific endonuclease or other more direct approaches to create nicks at sites of damage followed by quantitative Southern analysis and probing for specific genes. Initially, we used pyrimidine dimer specific endonuclease to detect pyrimidine dimers after UV irradiation. We now also use the bacterial enzyme ABC excinuclease to examine the DNA damage and repair of a number of adducts other than pyrimidine dimers in specific genes. We can detect gene specific alkylation damage by creating nicks via depurination and alkaline hydrolysis. In our assay for preferential repair, we compare the efficiency of repair in the DHFR gene to that in the 3′ flanking, non-coding region to the gene. In CHO cells, UV induced pyrimidine dimers are efficiently repaired from the active DHFR gene, but not from the inactive region. We have demonstrated that the 6–4 photoproducts are also preferentially repaired and that they are removed faster from the regions studied than pyrimidine dimers. Using similar approaches, we find that DNA adducts and crosslinks caused by cisplatinum are preferentially repaired in the active gene compared to the inactive regions and to the inactive c-fos oncogene. Also, nitrogen mustard and methylnitrosurea damage is preferentially repaired whereas dimethyisuiphate damage is not. NAAAF adducts do not appear to be preferentially repaired in this sytem.
In an attempt to correlate the repair events in specific regions with genomic translocations we have studied DNA damage and repair in the murine c-myc oncogene after UV damage and in the human c-myc after alkylation damage; these results will be discussed.
KeywordsChinese Hamster Ovary Cell Chinese Hamster Ovary Nitrogen Mustard Pyrimidine Dimer DHFR Gene
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- Bohr, V.A., Okumoto, D.S., Hanawalt, P.C. (1986). Survival of UV-irradiated mammalian cells correlates with efficient DNA repair in an essential gene. Proc Natl Acad Sci USA 83, 3830–3837.Google Scholar
- Bohr, V.A., Okumoto, D.S. (1988). Analysis of frequency of pyrimidine dimers in specific genomic sequences. In DNA repair: A laboratory manual. Edited by Friedberg EC, Hanawalt PC. Vol. 3, p 347. Marcel Dekker, New York.Google Scholar
- Ho, L., Bohr, V.A. and Hanawalt, P.C. (1989). Demethylation enhances removal of pyrimidine dimers from the overall genome and from specific DNA sequences in CHO cells. Mol. Cell. Biol. 9, 1594–1603.Google Scholar
- Mellon, I. and Hanawalt, P.C. (1989). Induction of the E. coli lactose operon selectively increases repair of its transcribed DNA strand. Nature 342, 95–98.Google Scholar
- Mitchell, D.L. (1988). The induction and repair of lesions produced by the photolysis of (6–4) photoproducts in normal and UV-hypersensitive human cells. Mutat Res 194, 227–37.Google Scholar
- Okumoto, D.S., and Bohr, V.A. (1987). DNA repair in the metallothionein gene increases with transcriptional activation. Nucl Acids Res 15, 10021–10031.Google Scholar
- Patrick, M.H., and Rahn, R.D. (1976). In Photochemistry and Photobiology of Nucleic Acids (S.Y. Wang, ed.) Vol II, pp 35–145. Acad. Press, N.Y..Google Scholar
- Potter, M. (1987). Mechanisms of B-cell neoplasia. Eds. Melchers, F. and Potter, M. Hoffman-La Roche and Co. Basle, Switzerland.Google Scholar
- Potter, M., Sanford, K.K., Parshad, R., Tarone, R.E., Price, F.M., Mock, B. and Huppi, K. (1988). Genes on chromosomes 1 and 4 in the mouse are associated with repair of radiation induced chromatin damage. Cenomics 2, 1–6.Google Scholar
- Tang, M-S., Bohr, V.A., Xang, X-S., Pierce, J. and Hanawalt, P.C. (1989). Quantitation of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using UVRABC nuclease. Journ. Biol. Chem. 264, 14455–14462.Google Scholar
- Thomas, D.C., Okumoto, D.S., Sancar, A. and Bohr, V.A. (1989). Preferential DNA repair of (6–4) photoproducts from the CHO DHFR gene. J. Biol. Chem 264, 18005–18010.Google Scholar
- Zdzienicka, M.Z., Roza, L., Westerveld, A., Bootsma, D. and Simons, J.W.I.M. (1987). Biological and biochemical consequences of the human repair gene ERCC-1 after transfection into a repair deficient CHO cell line. Mutat. Res., DNA Repair Reports 183, 69–75.Google Scholar