Dramatic changes in the ratio of homologous recombination to nonhomologous dma-end joining in oocytes and early embryos of xenopus laevis

Abstract

We have developed a versatile plasmid vector (pRecocr) for recombination studies. When linearized and introduced into the cells of interest, pReco-tr allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DMA-end joining (also termed end-to-end joining), the latter an example of illegitimate recombination processes. As a system we made use of stage VI oocytes and fertilized eggs of the African clawed frog Xenopus laevis, which were previously described to support homologous recombination and DMA-end joining, respectively. Extending these earlier findings, we show that oocytes yield >80% of the homologously recombined product, whereas in eggs a highly efficient DMA-end joining activity predominates (>95%). Both reactions, homologous recombination and DMA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DMA molecules are recircularized by DMA-end joining. With high amounts of injected DMA per fertilized egg, DMA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable. As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DMA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope with DNA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DMA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DNA into germ cells for gene targeting. Copyright ©by Walter de Gruyter & Co.