Homologous genetic recombination in Xenopus: mechanism and implications for gene manipulation.
Appropriately designed DNA substrates undergo very efficient homologous recombination after injection into the nuclei of Xenopus laevis oocytes. The requirements for this process are that the substrate be linear, that it have direct repeats to support recombination, and that these repeats be at or very near the molecular ends. Taking advantage of direct nuclear injection, the large amounts of DNA processed in a single oocyte, and the accessibility of recombination intermediates, we were able to analyze the mechanism of recombination in detail. Molecular ends are resected by a 5'-->3' exonuclease activity. When complementary sequences are exposed from two ends, they anneal. Continued 5'-->3' degradation removes the redundant strands; the 3' ends pair with their complements and can be extended by DNA polymerase to fill any gap left by the exonuclease. Joining of strands by DNA ligase completes the process. This mechanism is nonconservative, in that only one of the two original repeats is retained, and it has been dubbed single-strand annealing, or SSA. The capability for SSA accumulates during the later phases of oogenesis and persists into the egg. This pattern suggests that, like many activities of full-grown oocytes, SSA is stored for use during embryogenesis. The same or a very similar mechanism is prevalent in many other species, including bacteria, yeast, plants, and mammals, where it often provides the predominant mode of recombination of extrachromosomal DNA. Lessons learned about SSA are applicable to methods of gene manipulation. It is plausible that SSA has a normal function in the repair of double-strand breaks, but proof of this awaits identification of genes and enzymes uniquely involved in this style of recombination.
Carroll
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Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132, USA.
D000818:Animals; D002474:Cell-Free System; D004247:DNA / Q000235:genetics; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D004274:DNA, Recombinant / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors; D008845:Microinjections; D008957:Models, Genetic; D009865:Oocytes / Q000378:metabolism; D011995:Recombination, Genetic; D014162:Transfection; D014982:Xenopus laevis / Q000235:genetics*
D004274:DNA, Recombinant; D004247:DNA; D005090:Exodeoxyribonucleases; D011088:DNA Ligases