Efficient repair of DNA double-strand breaks in malignant cells with structural instability
| Autor: | Anna V. Roschke, Bridget P. Keenan, Kenneth Nakahara, Yue Cheng, Zhenhua Zhang, Peter D. Aplan |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
DNA double-strand break (DSB)
DNA Repair DNA repair Health Toxicology and Mutagenesis I-SceI Biology Antiviral Agents Thymidine Kinase Article Chromosome Painting chemistry.chemical_compound Plasmid Peptide Elongation Factor 1 Ovarian cancer Complementary DNA Genetics Tumor Cells Cultured Chromosomal rearrangement Humans DNA Breaks Double-Stranded Promoter Regions Genetic Molecular Biology Gene Ganciclovir In Situ Hybridization Fluorescence Etoposide Chromosome Aberrations Ovarian Neoplasms Expression vector Ovarian Neoplasms - genetics - pathology DNA Repair - genetics Molecular biology Antineoplastic Agents Phytogenic Antineoplastic Agents Phytogenic - pharmacology Blotting Southern Phosphotransferases (Alcohol Group Acceptor) chemistry Thymidine kinase Cinnamates Drug Resistance Neoplasm Female Hygromycin B DNA Plasmids |
| Popis: | Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1α) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1α promoter from the TK gene, or deletion of either the EF1α promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR. link_to_OA_fulltext |
| Databáze: | OpenAIRE |
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