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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumHomologous DNA recombination in vertebrate cellsEiichiro Sonoda*†, Minoru Takata*‡, Yukiko M.Yamashita*, Ciaran Morrison§, and Shunichi Takeda*†¶ *Department of Radiation Genetics, Faculty of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606–8501, Japan; †CREST (Core Research for Evolutional Science and Technology), JST (Japan Science and Technology), Kawaguchi, Japan; ‡Department of Immunology, Kawasaki Medical School, Kurashiki, Japan; and §Institute of Cell and Molecular Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, United Kingdom The RAD52 epistasis group genes are involved in homologous DNA recombination, and their primary structures are conserved from yeast to humans. Although biochemical studies have suggested that the fundamental mechanism of homologous DNA recombination is conserved from yeast to mammals, recent studies of vertebrate cells deficient in genes of the RAD52 epistasis group reveal that the role of each protein is not necessarily the same as that of the corresponding yeast gene product. This review addresses the roles and mechanisms of homologous recombination-mediated repair with a special emphasis on differences between yeast and vertebrate cells. DT40 reverse genetic study | double-strand break repair | Rad 51 family Chicken DT40 Cells as an Experimental System to Analyze Homologous Recombination (HR)Chicken B lymphocyte precursors diversify the variable segments of their Ig genes not only by site-specific V(D)J DNA recombination, but also by a HR process, Ig gene conversion, which occurs in the bursa of Fabricius (recently reviewed in ref. 1). Mature chicken B lymphocytes are also capable of undergoing Ig gene conversion in splenic germinal centers on antigenic stimulation (2). A chicken B lymphocyte line, DT40, transformed with an avian leukosis virus, continuously undergoes Ig gene conversion during in vitro culture (3, 4). Remarkably, targeted integration occurs in these cells with efficiencies that are orders of magnitude higher than those observed in mammalian cells (5). This integration occurs at all loci analyzed including silent loci such as the ovalbumin and α-crystalin loci. Efficient gene targeting is demonstrated in all chicken B lymphocyte lines analyzed, including another avian leukosis virus-transformed cell line, RP9, and a v-rel-transformed cell line 27L2, suggesting that this extraordinary capability might be shared by even some ex vivo chicken B lymphocytes (5). The molecular mechanism responsible for the high targeting efficiencies in chicken B lymphocyte lines is not clear. Conceivably, a common molecule may be responsible not only for Ig gene conversion but also for enhancing gene targeting efficiencies, because both these processes are mediated by HR and are observed only in chicken B lymphocytes but not in chicken non-B cells or mammalian cell lines. Although we have found some molecules to be required for efficient gene targeting in DT40 cells (Table 1), none of them appear to account for the high levels of gene targeting because they are expressed both in DT40 cells and the chicken non-B cell lines. Besides efficient gene targeting, DT40 cells possess a number of advantages as a tool for reverse genetic studies. First, the relatively invariant character of DT40 cells, during extended periods of cell culture, allows for the performance of sequential gene targeting of up to three genes in a single cell using seven different selection marker genes. Because some DNA repair pathways are complementary to each other, cells deficient in two repair pathways often exhibit an extremely severe phenotype when compared with cells deficient in either pathway alone (10). Using this reasoning we have been able to investigate distinct as well as overlapping roles of independent repair pathways by knocking out multiple genes involved in DNA repair in DT40 cells. Second, the extremely rapid growth rate of DT40 cells, with a doubling time of 8–10 h, makes it easy to perform phenotypic analysis. Third, the absence of functional p53 in DT40 cells offers an additional advantage in the analysis of mutant cells exhibiting genome instability (15). DNA lesions in such mutant cells would elevate the level of p53 product, leading to the decrease in the cloning efficiency of cells, significantly reducing the growth rate, and inducing apoptosis. Although HR-deficient yeast mutants are viable, some HR-deficient vertebrate cells show impaired proliferation or even lethality (6, 13) (Table 1). Therefore, we have investigated the effect of HR deficiency in vertebrate cells by generating conditionally mutant cells. Three methods have been used successfully for conditionally suppressing the expression of genes in DT40 cells. As shown in Fig. 1 for RAD51, the structural and functional homologue of Escherichia coli RecA, we generated conditionally null DT40 clones that express a human transgene under the control of a tetracyclin-repressible promoter (6). Although the promoter activity was suppressed by more than 100-fold upon the addition of tetracyclin, possible effects of leaky expression cannot be excluded (16). One way to overcome this disadvantage is to use a chimeric Cre recombinase. The Cre recombinase recognizes loxP sequences and deletes or inverts sequences between two loxP sites depending on the relative orientation of these two loxP sequences. The chimeric Cre recombinase carries a mutated hormone-binding domain of the murine estrogen receptor (17), which binds the antagonist 4-hydroxytamoxifen (OH-TAM). Upon the addition of OH-TAM to the culture media, the chimeric Cre recombinase is translocated into the nucleus where it recognizes loxP sites and recombines the DNA to delete the gene of interest. Cre-mediated recombination works efficiently in DT40 cells; virtually all of the genes flanked by two loxP sequences were deleted within 24 h after the addition of OH-TAM (unpublished work). Although this system allows us to completely inhibit the expression of a gene of interest, Cre-mediated recombination does not occur in a synchronous manner in a population of cells, as observed in the tet repressible promoter system. A third method of generating conditionally mutant clones uses temperature sensitive (ts) mutant genes. The physiological body temperature of the chicken ranges from 40.9°C to 41.9°C; the cell culture temperature can vary from 34°C to as high as 43°C without loss of viability. In a procedure designed to generate ts mutants of an essential gene, such as that encoding the kinet This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: HR, homologous recombination; ts, temperature sensitive; DSB, double-strand break; NHEJ, nonhomologous end-joining; ES, embryonic stem; SCE, sister chromatid exchange; IR, ionizing radiation.
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