Strand Invasion and DNA Synthesis From the Two 3′ Ends of a Double-Strand Break in Mammalian Cells

Abstract

Abstract Analysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion (“ends-in”) gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3′ vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3′ ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.