Transcription-coupled and transcription-independent repair of cyclobutane pyrimidine dimers in the dihydrofolate reductase gene.
Using a ligation-mediated polymerase chain reaction technique, we have mapped the repair of ultraviolet light-induced cyclobutane pyrimidine dimers (CPDs) at the nucleotide level in exons 1, 2, and 5 of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells. We found that CPDs are preferentially repaired in the transcribed strand (T strand) and that the order of repair efficiency is exon 1 > exon 2 > exon 5. In the cells with a deletion of the DHFR gene encompassing the promoter region and the first four exons, CPDs are not repaired in the T strand of the residual DHFR gene. These results substantiate the idea that the preferential repair of CPDs in the T strand is transcription dependent. However, in the wild type gene we have found that CPDs are efficiently repaired in the nontranscribed strand (NT strand) of exon 1 but not in the NT strand of exons 2 and 5. Probing the chromatin structure of exons 1, 2, and 5 of the DHFR gene with micrococcal nuclease revealed that the exon 1 region is much more sensitive to micrococcal nuclease digestion than the exon 2 and exon 5 regions, suggesting that the chromatin structure in the exon 1 region is much more open. These results suggest that, although preferential repair of the T strand of the DHFR gene is transcription dependent, repair of the NT strand is greatly affected by chromatin structure.
Hu
Wenwei
W
Department of Environmental Medicine, Pathology, and Medicine, New York University School of Medicine, Tuxedo, New York 10987, USA.
Feng
Zhaohui
Z
Chasin
Lawrence A
LA
Tang
Moon-shong
MS
D000818:Animals; D016466:CHO Cells; D002843:Chromatin / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000528:radiation effects; D004260:DNA Repair; D005091:Exons / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002843:Chromatin; D011740:Pyrimidine Dimers; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase