Double-strand gap repair results in homologous recombination in mouse L cells

Abstract

Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event.