Translesion DNA synthesis in the dihydrofolate reductase domain of UV-irradiated CHO cells.
The studies that document the coupling of strand-specific DNA repair to transcription of active genes exclude replicated DNA from the analysis. Yet cyclobutane pyrimidine dimers (CPD) induced by ultraviolet light (UV) persist in most of the genome in surviving Chinese hamster ovary (CHO) cells. The mechanisms that allow DNA replication to occur in the presence of damaged templates are poorly understood. We have investigated the distribution of CPD in the dihydrofolate reductase gene (DHFR) domain in replicated DNA. CHO B11 cells were incubated in the presence of BrdUrd after UV irradiation; the replicated DNA was separated from the unreplicated DNA by isopycnic sedimentation in CsCl, and then the parental and daughter strands were resolved in alkaline CsCl. We determined the fraction of a 14-kb KpnI fragment of the DHFR gene that was resistant to digestion by T4 endonuclease V, a CPD-specific enzyme. In both parental and unreplicated DNA, approximately 80% of the CPD were removed from the transcribed strands while approximately 20% were removed from the nontranscribed strands of DHFR within 24 h. In a 15-kb KpnI fragment that contains an origin of replication and is located approximately 15 kb downstream of DHFR, we found very low repair levels, whether it had been replicated or not. We detected no CPD in the daughter strands of either fragment analyzed. These results suggest that the replication forks can move through the damaged DNA in the absence of significant levels of repair or strand exchange and that the repair of CPD is not affected by replication in these cells.
Spivak
G
G
Department of Biological Sciences, Stanford University, California 94305-5020.
Hanawalt
P C
PC
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication / Q000528:radiation effects*; D005784:Gene Amplification; D016678:Genome; D008957:Models, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase