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 dimethylsulphate damage is not. NAAAF adducts do not appear to be preferentially repaired in this system.(ABSTRACT TRUNCATED AT 250 WORDS)
Bohr
V A
VA
Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892.
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D016259:Genes, myc / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D009369:Neoplasms / Q000209:etiology / Q000235:genetics; D014466:Ultraviolet Rays
D000970:Antineoplastic Agents; D029968:Escherichia coli Proteins; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC