Induction of mutations at the thymidine kinase locus in CHO cells by restriction endonucleases.
Induced mutation frequencies were measured at the tk locus (encoding for the enzyme thymidine kinase) following treatment of Chinese hamster ovary cells (CHO KI) with two restriction endonucleases (REs), PvuII and EcoRI, which generate 'blunt-ended' and 'cohesive-ended' DNA double-strand breaks (dsb), respectively. Electroporation was used to introduce these enzymes into the cells. Restriction endonucleases generating blunt-ended dsb have been shown to mimic the action of ionising radiation in causing chromosome aberrations, cell killing, mutations and oncogenic transformation. Here we show that the tk locus, PvuII induced an approximately 11-fold higher mutation frequency than EcoRI at the same enzyme concentrations. There are four PvuII and six EcoRI restriction sites in the Chinese hamster thymidine kinase gene. Hence the higher mutation induction by PvuII, despite the lower number of restriction sites than EcoRI in the tk gene, suggests that blunt-ended dsb represent more effective and critical mutagenic DNA lesions than the cohesive-ended type. In this respect, our results are similar to those we obtained previously for chromosomal aberrations and for cell killing. Results from the present study suggest that mutations could arise from unrepaired or misrepaired dsb possibly via induction of chromosomal deletions or stable exchanges between chromosomes.
Singh
B
B
Department of Biology and Pre-clinical Medicine, University of St Andrews, Fife, UK.
Bryant
P E
PE
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D015246:Deoxyribonuclease EcoRI / Q000494:pharmacology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000494:pharmacology*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects* / Q000528:radiation effects; D016296:Mutagenesis; D010053:Ovary; D013937:Thymidine Kinase / Q000235:genetics*
D013937:Thymidine Kinase; D015246:Deoxyribonuclease EcoRI; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific