Ataxia telangiectasia: an investigation of the repair defect in the cell line AT5BIVA by plasmid reconstitution.
The ataxia telangiectasia cell line, AT5BIVA, exhibited low repair fidelity measured by the reconstitution of transfected linear plasmid. This assay involves transfecting a linear plasmid containing two selectable marker genes: one gene (neo) is undamaged and marks transfection and the other gene (gpt) is cleaved to test functional repair. The proportion of transfected cells which have a functionally intact gpt gene gives a measure of repair fidelity. Southern analysis of individual transfected clones showed that integrated plasmids in AT5BIVA had a high frequency of sequence rearrangement. Blunt or staggered-ended termini of a linear plasmid did not determine the type of misrepair. A variety of sizes of deletions and sequence insertions were found at and around the cleavage site. Loss of intact sequence occurred similarly following transfection by linear or circular plasmid (misrepair or rearrangement error). This suggests that the action of excess exonuclease activity upon, or lack of protection of, exposed DNA termini is not the sole mechanism of misrepair. Erroneous rearrangement of circular plasmid could involve any location along the plasmid. Rearrangement of transfected circular plasmid occurred in multiple copies of the same abnormal size, suggesting that error-prone recombination rather than degradation of presumed nicked circular plasmid was the underlying mechanism. It is hypothesized that misrepair in ataxia-telangiectasia arises by error-prone recombination.
Powell
S
S
Radiotherapy Research Unit, Institute of Cancer Research, Sutton, Surrey, UK.
Whitaker
S
S
Peacock
J
J
McMillan
T
T
D001260:Ataxia Telangiectasia / Q000235:genetics*; D015139:Blotting, Southern; D002461:Cell Line, Transformed; D003001:Cloning, Molecular; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific; D005347:Fibroblasts; D006801:Humans; D010957:Plasmids; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014162:Transfection
D015252:Deoxyribonucleases, Type II Site-Specific; C027440:GATATC-specific type II deoxyribonucleases; C029312:GGTACC-specific type II deoxyribonucleases