Homologous recombination and DNA-end joining reactions in zygotes and early embryos of zebrafish (Danio rerio) and Drosophila melanogaster.

A linear DNA with partial sequence redundancy can be recircularized in cells by either nonhomologous end joining (NEJ) or by homologous recombination (HR). We have studied the relative contributions of these processes in zygotes or early embryos of species that serve as model organisms for developmental genetics. Thus, we have microinjected a linearized plasmid substrate into zygotes of zebrafish (Danio rerio) or into the posterior end of Drosophila melanogaster early embryos before pole cell formation. Similar to the situation observed previously in Xenopus zygotes/early embryos, we detected a large preponderance of DNA-end joining over homologous recombination. A comparison of end-joined junctions revealed that from the three species tested, zebrafish introduced the least number of sequence distortions upon DNA-end joining, while Drosophila produced the largest deletions (average 14 bp) with occasional nucleotide patch insertions, reminiscent of the N nucleotides at V(D)J junctions in mammalian immune receptor genes. Double-strand gap repair by homologous sequences ('homologous recombination') involving a bimolecular reaction was readily detectable in both zebrafish and Drosophila. This involved specifically designed recombination substrates consisting of a mutagenized linear plasmid and DNA fragments carrying the wild-type sequence. Our results show that the basic machinery for homologous recombination is present at early developmental stages of these two genetic model organisms. However, it seems that for any experimental exploitation, such as targeted gene disruption, one would have to inhibit or bypass the overwhelming DNA-end joining activity.

Dramatic changes in the ratio of homologous recombination to nonhomologous DNA-end joining in oocytes and early embryos of Xenopus laevis.

We have developed a versatile plasmid vector (pReco-sigma) for recombination studies. When linearized and introduced into the cells of interest, pReco-sigma allows the simultaneous determination of the relative frequencies of homologous recombination versus nonhomologous DNA-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 DNA-end joining, respectively. Extending these earlier findings, we show that oocytes yield > 80% of the homologously recombined product, whereas in eggs a highly efficient DNA-end joining activity predominates (> 95%). Both reactions, homologous recombination and DNA-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 DNA molecules are recircularized by DNA-end joining. With high amounts of injected DNA per fertilized egg, DNA-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 DNA-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 DNA 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.