Homologous recombination in mammalian cells.

Mouse LTKcells were transfected with a pair of defective Herpes simplex virus thymidine kinase ( t k ) genes. One tk gene had an 8-bp insertion mutation while the second gene had a 100-bp inversion. Extrachromosomal homologous recombination leading to the reconstruction of a functional tk gene was monitored by selecting for tk positive cells using medium supplemented with hypoxanthine/ aminopterin/thymidine. To assess whether the search for homology may be a rate-limiting step of recombination, we asked whether the presence of an excess number of copies of a tk gene possessing both the insertion and inversion mutations could inhibit recombination between the singly mutated tk genes. Effective competitive inhibition would require that homology searching (homologous pairing) occur rapidly and efficiently. We cotransfected plasmid constructs containing the singly mutated genes in the presence or absence of competitor sequences in various combinations of linear or circular forms. We observed effective inhibition by the competitor DNA in six of the seven combinations studied. A lack of inhibition was observed only when the insertion mutant gene was cleaved within the insertion mutation and cotransfected with the two other molecules in circular form. Additional experiments suggested that homologous interactions between two DNA sequences may compete in trans with recombination between two other sequences. We conclude that homology searching is not a rate-limiting step of extrachromosomal recombination in mammalian cells. Additionally, we speculate that a limiting factor is involved in a recombination step following homologous pairing and has a high affinity for DNA termini. I N recent years, there have been many studies into the processes of homologous recombination in eukaryotes but many fundamental questions remain unanswered. At least three modes of homologous recombination events have been studied in mammalian cells. These events can be classified as extrachromosomal, intrachromosomal and targeted homologous recombination (“gene targeting”) (reviewed in BOLLAC et al. 1989). Studies have suggested that there may be some mechanistic differences between these three recombination modes. For example, extrachromosomal recombination in mammalian cells is less sensitive to scattered heterologies than is intrachromosomal recombination between direct repeats (WALDMAN and LISKAY 1987). Further, gene targeting appears to be less sensitive to blocks of heterology embedded within homologous sequences than is intrachromosomal recombination (MANSOUR et al. 1990; reviewed in WALDMAN 1992). All three modes of homologous recombination, however, share a fundamental similarity in that they all can be described as an exchange of nucleotides between similar DNA sequences. It is therefore reasonable to assume that the mechanisms of the three recombination modes are not entirely distinct; studies have in fact illuminated several similarities. All modes of recombination Genetics 136 597-605 (February, 1994) are sensitive to the overall length of homology shared by the recombining sequences (BOLLAG et al. 1989; HASTY et al. 1991; LISKAY et al. 1987; RUBNITZ and SUBRAMANI 1984; THOMAS and CAPECCHI 1987; WALDMAN 1992). Best estimates are that extrachromosomal (RUBNITZ and SUBRAMANI 1984) as well as intrachromosomal (LISKAY et al. 1987; WALDMAN and LISKAY 1988) recombination require about 150-200 bp of homology in order to proceed efficiently. A strong homology length dependence of targeting has also been demonstrated by at least two groups (HASTY et al. 1991; THOMAS and CAPECCHI 1987). Additionally, both extrachromosomal recombination and gene targeting can be stimulated by the appropriate placement of breaks in a transfected sequence (BOLLAG et al. 1989; WALDMAN 1992). Finally, we have recently shown that the positioning of an SV40 promoter/enhancer sequence on a recombination substrate could exert qualitatively similar effects on both extraand intrachromosomal recombination (YANG and WALDMAN 1992), suggesting some overlap in the mechanisms of these recombination pathways. A basic issue regarding any recombination pathway is the question of what constitutes the rate-limiting step of the recombination process. It is clear that mammalian cells do not have an unlimited capacity to