Intrachromosomal homologous recombination in human cells which differ in nucleotide excision-repair capacity

Abstract

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 (H tk) 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 3er 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 H tk genes had become wild-type, i.e., XhoI-resistant. 90% of the recombinants retained the H tk gene duplication, consistent with non-reciprocal transfer of genetic information, i.e., gene conversion. The rest contained a single, wild-type H tk 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.