Gene recombination in X-ray-sensitive hamster cells.

Abstract

Recombination was measured in Chinese hamster ovary (CHO-Kl) cells and in the X-ray-sensitive mutants xrsl and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the dilTerent cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-Kl and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-Kl, but variation In microbes, a genetic link has been established between ionizing-radiation resistance and recombination proficiency; prominent examples of the genes involved are found in the RecF pathway of Escherichia coli (41) and in the RAD52 group of Saccharomyces cerevisiae (14). Additionally, mutation of such genes often affects the ability to repair DNA double-strand breaks. Thus, it has been hypothesised that double-strand breaks induced by ionizing radiation are rejoined by a process of recombination between homologous chromosomal regions (24).To investigate the relationship between ionizing-radiation resistance and recombination in mammalian cells, we have examined the recombination proficiencies of wild-type and X-ray-sensitive (xrs) hamster cells. The xrs mutants were isolated from a Chinese hamster ovary (CHO) cell line by Jeggo and Kemp (10). These mutants lack the ability to recover from potentially lethal radiation damage (38) and show reduced repair of DNA double-strand breaks (11, 43). The two mutants selected for this study, xrsl and xrs7, belong to the same complementation group (9) and differ only slightly in their responses to ionizing radiations (10, 38), but show substantially different responses to other genotoxic agents (10).The study of recombination in mammalian cells has been facilitated in the last few years by the use of virus-and plasmid-based gene transfer methods. It has been shown that mammalian cells have the enzymatic processes necessary for both homologous and nonhomologous recombination of introduced DNA (6,12,23,27,31,32,34,39,40,44). We have attempted to apply these methods quantitatively by carefully controlling DNA transfer conditions to determine plasmid recombination efficiencies in the parent and mutant CHO cells. Additionally we have followed up the observation, made initially for plasmid-chromosome recombination in S. cerevisiae (21), that a double-strand break in regions of * Corresponding author. homology greatly stimulates recombination. While our experiments were in progress on plasmid-plasmid recombination in CHO cells, it was reported that double-strand breaks are...

Chromosomal double-strand break repair in Ku80-deficient cells.

“…At low DNA concentrations, little or no difference is observed between wild-type and mutant cell lines. At high DNA concentrations, as many as 5-to 10-fold fewer random integrants are obtained from xrs-6 cells and other members of the IR complementation group 5, as measured by selectable drug marker expression (27,28). These results, combined with the proficiency of these cells to undergo DSB-promoted recombination events, suggest that Ku deficient cell lines may have more desirable ratios of homologous to nonhomologous integrations in gene targeting studies, at least under some transfection conditions.…”

“…In our assays, electroporatedxrs-6 cells exhibited no deficiency in the integration of the control plasmid pMClneo, suggesting that there may be mechanistic differences between chromosome end-joining and random plasmid integration. Previous studies in xrs cells using calcium phosphate transfection have reported a defect in plasmid integration that is dependent on the concentration of the transfected DNA (27,28). At low DNA concentrations, little or no difference is observed between wild-type and mutant cell lines.…”

Manipulating the mammalian genome by homologous recombination

“…Observations on plasmid integration in Ku80p-deficient cells also implicate an additional mechanism for random integration that becomes saturated at high DNA concentrations (128,129). At low DNA concentrations little difference is observed between wild-type and Ku80p-deficient cells; however, at high DNA concentrations random integration is 5-to 10-fold lower in Ku80p-deficient cells (128,129).…”

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining

“…Each plasmid carries a copy of the bacterial xgprt gene, which has been inactivated either by a 5Ј or 3Ј deletion (p⌬2-puro or p⌬3, respectively). Neither of the xgprt mutations are revertible, and only HR, via either gene conversion or gene crossover between these two mutant copies can reconstitute a functional xgprt gene, thereby conferring resistance to XHATM drug selection in cell culture (14,33). For cells without wt BRCA2, a mean homology-mediated chromosomal-targeting frequency of Fig.…”