Intrachromosomal homologous recombination in human cells which differ in nucleotide excision-repair capacity.
To examine the mechanism of recombination and the role of DNA repair in this process, we transfected a plasmid carrying duplicated copies of the Herpes simplex virus I thymidine kinase (Htk) gene, each containing an 8 bp XhoI site inserted in a unique site and with the neo coding for geneticin resistance located between them, into tk-deficient human cell lines which differ in their ability to carry out nucleotide excision repair. One parental cell line has a normal level of repair activity; the second has an intermediate level, and the third has virtually no repair activity. Several geneticin-resistant transfectant cell strains from each parental line were isolated and assayed for the ability to undergo productive recombination giving rise to tk+ cells. Approximately 25% of them could do so. Southern blot analysis of these transfectants indicated that the majority contained a single copy, or at most, two copies of the plasmid integrated into the chromosome. Fluctuation analysis tests to determine the rate of spontaneous recombination (events per 10(6) cells per cell generation) in the various cell strains showed that the rates ranged from 0.15 to 4.1. The mean rate for the cell strains derived from the repair-deficient cell line was 3.6; for those derived from the cells with an intermediate rate, it was 0.8; and for those with a normal rate of excision repair, it was 0.9. Southern blot analysis of tk+ recombinants showed that in all cases, one of the Htk genes had become wild-type, i.e., XhoI-resistant. 90% of the recombinants retained the Htk gene duplication, consistent with non-reciprocal transfer of genetic information, i.e., gene conversion. The rest contained a single, wild-type Htk gene, consistent with a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. These cell strains will be useful for investigating the role of DNA damage and repair in homologous recombination.
Bhattacharyya
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Department of Microbiology, Michigan State University, East Lansing 48824-1316.
Maher
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VM
McCormick
J J
JJ
D015139:Blotting, Southern; D002470:Cell Survival / Q000528:radiation effects; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair; D004351:Drug Resistance / Q000235:genetics; D005814:Genes, Viral; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D003470:Culture Media; D005839:Gentamicins; D004247:DNA; C010680:antibiotic G 418; D013937:Thymidine Kinase