Intrachromosomal Recombination Between Well-Separated, Homologous Sequences in Mammalian Cells

Abstract

Abstract In the present study, we investigated intrachromosomal homologous recombination in a murine hybridoma in which the recipient for recombination, the haploid, endogenous chromosomal immunoglobulin μ-gene bearing a mutation in the constant (Cμ) region, was separated from the integrated single copy wild-type donor Cμ region by ∼1 Mb along the hybridoma chromosome. Homologous recombination between the donor and recipient Cμ region occurred with high frequency, correcting the mutant chromosomal μ-gene in the hybridoma. This enabled recombinant hybridomas to synthesize normal IgM and to be detected as plaque-forming cells (PFC). Characterization of the recombinants revealed that they could be placed into three distinct classes. The generation of the class I recombinants was consistent with a simple unequal sister chromatid exchange (USCE) between the donor and recipient Cμ region, as they contained the three Cμ-bearing fragments expected from this recombination, the original donor Cμ region along with both products of the single reciprocal crossover. However, a simple mechanism of homologous recombination was not sufficient in explaining the more complex Cμ region structures characterizing the class II and class III recombinants. To explain these recombinants, a model is proposed in which unequal pairing between the donor and recipient Cμ regions located on sister chromatids resulted in two crossover events. One crossover resulted in the deletion of sequences from one chromatid forming a DNA circle, which then integrated into the sister chromatid by a second reciprocal crossover.