Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.
Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.
Puchta
H
H
Friedrich Miescher-Institut, Basel, Switzerland.
Dujon
B
B
Hohn
B
B
D001483:Base Sequence; D003001:Cloning, Molecular; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D018390:Gene Targeting; D018745:Genome, Plant; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics* / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012231:Rhizobium / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D014170:Transformation, Genetic
D018744:DNA, Plant; D029701:Saccharomyces cerevisiae Proteins; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific