Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.
Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.
Puchta
H
H
Friedrich Miescher-Institut, Basel, Switzerland.
Dujon
B
B
Hohn
B
B
D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D005813:Genes, Synthetic; D005966:Glucuronidase; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014026:Nicotiana / Q000235:genetics*; D014162:Transfection
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D005966:Glucuronidase