A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells.
Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.
D000818:Animals; D016590:Aphidicolin; D002453:Cell Cycle / Q000187:drug effects; D002472:Cell Transformation, Viral; D002945:Cisplatin / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D004260:DNA Repair / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004273:DNA, Neoplasm / Q000378:metabolism; D004224:Diterpenes / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects; D006801:Humans; D007939:Leukemia L1210; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D013539:Simian virus 40; D014983:Xeroderma Pigmentosum
D003432:Cross-Linking Reagents; D003470:Culture Media; D004273:DNA, Neoplasm; D004224:Diterpenes; C034697:transplatin; D016590:Aphidicolin; D003513:Cycloheximide; D002945:Cisplatin