Repair of N-methylpurines in specific DNA sequences in Chinese hamster ovary cells: absence of strand specificity in the dihydrofolate reductase gene.
We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.
Scicchitano
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DA
Department of Biological Sciences, Stanford University, CA 94305-5020.
Hanawalt
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PC
D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004249:DNA Damage; D015342:DNA Probes; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D005091:Exons; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D013463:Sulfuric Acid Esters / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D015342:DNA Probes; D015347:RNA Probes; D013463:Sulfuric Acid Esters; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases; D000225:Adenine; C007482:dimethyl sulfate; D004121:Dimethyl Sulfoxide