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Recent progress with the DNA repair mutants of Chinese hamster ovary cells
L H Thompson1, E P Salazar, K W Brookman
1Biomedical Sciences Division, Lawrence Livermore National Laboratory, CA 94550.
Abstract:
Repair-deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant, which is noted for its very high sister-chromatid exchange frequencies. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to chlorodeoxyuridine (CldUrd). After EcoRI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25-30 kilobases (kb) was present. Since the DNA molecules used to produce these transformants were sheared to less than 50 kb in size, the correcting gene should be small enough to clone in a cosmid vector. Using drug-resistance markers to select for hybrids after fusion, we have done complementation experiments with ultraviolet light (u.v.)-sensitive mutants and have identified a sixth complementation group, line UV61. Additionally, CHO mutants UV27-1 and MMC-2, isolated in other laboratories, were found to belong to UV group 3, which is represented by line UV24. To study the behaviour of transfected DNA molecules in repair-deficient cells, we treated plasmid pSV2gpt with either u.v. radiation or cis-diamminedichloroplatinum(II) (cis-DDP) and introduced the damaged DNA into normal CHO cells (AA8) and mutants UV4 and UV5. Unrepaired damage to the plasmid was indicated by loss of colony-forming ability of the transfected cells in selective medium containing mycophenolic acid. With u.v. damage, the differential survival of the cell lines was similar to that seen when whole cells are treated with u.v. However, with cis-DDP damage, mutant UV4 did not exhibit the extreme hypersensitivity (50-fold) that occurs when cells are treated. This result suggests that UV4 cells may be able to repair cross-links in transfected DNA.
Insights
Researchers identified a human DNA sequence that corrects DNA repair defects in Chinese hamster ovary (CHO) cells, aiding the study of DNA repair and mutagenesis mechanisms. This discovery advances understanding of genetic repair processes.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Repair-deficient Chinese hamster ovary (CHO) cell mutants are crucial for identifying human genes involved in DNA repair and mutagenesis.
- The EM9 mutant exhibits exceptionally high sister-chromatid exchange frequencies, making it a valuable model for repair studies.
Purpose of the Study:
- To identify human DNA sequences that can correct DNA repair defects in CHO cell mutants.
- To investigate the mechanisms of DNA repair and mutagenesis using genetically modified CHO cells.
- To complement and classify ultraviolet light (u.v.)-sensitive CHO mutants.
Main Methods:
- DNA transformation of EM9 mutant CHO cells with human DNA, followed by selection for chlorodeoxyuridine (CldUrd) resistance.
- Southern transfer and hybridization techniques using Alu family sequences to identify integrated human DNA fragments.
- Cell fusion and drug-resistance marker selection for complementation experiments with u.v.-sensitive mutants.
- Transfection of damaged plasmid DNA (u.v. or cis-diamminedichloroplatinum(II)) into normal and mutant CHO cells to assess repair capabilities.
Main Results:
- A human DNA sequence was identified in five independent transformants that conferred resistance to CldUrd.
- A common 25-30 kilobase (kb) DNA fragment was detected in transformants, suggesting a gene small enough for cosmid cloning.
- Complementation experiments identified a sixth complementation group (UV61) among u.v.-sensitive mutants.
- CHO mutants UV27-1 and MMC-2 were classified into UV group 3.
- Mutant UV4 showed less hypersensitivity to cis-diamminedichloroplatinum(II) (cis-DDP) damage in transfected DNA compared to whole-cell treatment, suggesting potential repair of cross-links.
Conclusions:
- A human DNA sequence capable of correcting specific DNA repair defects in CHO cells has been identified.
- The study advances the understanding of DNA repair pathways and the genetic basis of mutagenesis.
- The findings suggest that the UV4 mutant may possess a mechanism for repairing DNA cross-links introduced into transfected plasmids.