The search for homology does not limit the rate of extrachromosomal homologous recombination in mammalian cells.
Mouse LTK- cells were transfected with a pair of defective Herpes simplex virus thymidine kinase (tk) 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.
Waldman
A S
AS
Department of Biological Sciences, University of South Carolina, Columbia 29208.
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D007446:Chromosome Inversion; D004274:DNA, Recombinant / Q000235:genetics; D005111:Extrachromosomal Inheritance / Q000235:genetics*; D007739:L Cells; D008322:Mammals / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D004274:DNA, Recombinant; D014764:Viral Proteins; D013937:Thymidine Kinase