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Two Different but Related Mechanisms Are Used in Plants for the Repair Of Proc. Natl. Acad. Sci. USA Vol. 93, pp. 5055–5060, May 1996 Genetics Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination (site-specific integrationyone-sided invasionyI-Sce IyDNA polymerase slippageyAgrobacterium) HOLGER PUCHTA*†‡,BERNARD DUJON§, AND BARBARA HOHN* *Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland; †Institut fu¨r Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany; and §Unite´deGe´ne´tique Mole´culaire des Levures, De´partement des Biotechnologies (Unite´ de Recherche Associe´e 1149yCentre National de la Recherche Scientifique), Institut Pasteur, 25 rue du Dr. Roux, F-75724 Paris Cedex 15, France Communicated by Diter von Wettstein, Carlsberg Laboratory, Gamle Carlsberg, Copenhagen, Denmark, January 2, 1996 (received for review September 1, 1995) ABSTRACT Genomic double-strand breaks (DSBs) are mologous recombination. Factors that introduce unspecific key intermediates in recombination reactions of living organ- DSBs in DNA, such as x-rays (15, 16) or methyl methanesul- isms. We studied the repair of genomic DSBs by homologous fonate (17), were shown to enhance intrachromosomal homol- sequences in plants. Tobacco plants containing a site for the ogous recombination in plants. Excision of transposable ele- highly specific restriction enzyme I-Sce I were cotransformed ments was correlated with increased frequencies of recombi- with Agrobacterium strains carrying sequences homologous to nation at the donor site between repeats flanking the element the transgene locus and, separately, containing the gene (18, 19) or between repeats at ectopic sites (A. Levy, personal coding for the enzyme. We show that the induction of a DSB communication). can increase the frequency of homologous recombination at a We demonstrated that expression of the I-Sce I gene led to specific locus by up to two orders of magnitude. Analysis of the specific DSBs in vivo in DNA molecules transfected into plant recombination products demonstrates that a DSB can be protoplasts (10). As a consequence extrachromosomal homol- repaired via homologous recombination by at least two dif- ogous recombination was induced. This reaction proceeds ferent but related pathways. In the major pathway, homologies mainly via single-strand annealing and thus seems to be on both sides of the DSB are used, analogous to the conser- different from recombination reactions in the chromosome (as vative DSB repair model originally proposed for meiotic discussed in ref. 20). In the present communication we ana- recombination in yeast. Homologous recombination of the lyzed the mechanism by which DSBs at specific sites in the minor pathway is restricted to one side of the DSB as described plant genome are repaired with homologous sequence infor- by the nonconservative one-sided invasion model. The se- mation, and we tested whether induction of DSBs is correlated quence of the recombination partners was absolutely con- with an increase in recombination frequencies. served in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage MATERIALS AND METHODS during the copy process. The induction of DSB breaks to Cloning Procedures. Construction of the I-Sce I expression enhance homologous recombination can be applied for a 1 variety of approaches of plant genome manipulation. vector. The plasmid p35SISceI (10) containing an artificial I-Sce I open reading frame (ORF) under the control of the cauliflower 35S promoter and the cauliflower terminator was Double-strand breaks (DSBs) play an important role in ho- cut with BamHI and the ORF containing fragment was mologous recombination in eukaryotes (1–3). Recently, ex- inserted in the unique BamHI site of the binary vector perimental evidence that meiotic recombination in yeast is pCGN1589 (21) to result in the plasmid pCISceI. initiated in most, if not all, cases by DSBs was supplied (4). Construction of the target site. Into the EcoRV site of the Mitotic gene conversion in mating type switching in yeast is plasmid pAF100 (22), the Xho I–Acc65I-cut, blunt-ended DNA initiated by a DSB at the recognition site of HO endonuclease fragment of the plasmid pAH5 (23) carrying the C-terminal half (for review, see ref. 5). Knowledge on the importance of DSBs of an artificial, intron-containing kanamycin resistance gene was for homologous recombination has been employed to induce cloned. The resultant plasmid pAFAH was cut by Acc65I, and the recombination reactions. Cleavage of the HO nuclease recog- specific fragment was ligated into the Acc65I-digested binary nition site by induction of the HO nuclease expression has been vector pCH (24), resulting in the plasmid pTS. used to enhance intra- and intermolecular recombination in Production of the repair construct. A specific polylinker with yeast cells (for review, see ref. 6). Also the yeast mitochondrial the sequence 59-pGAATTCGTTA ACGAGCTCTC TAGA- I-Sce I endonuclease (7), which has an 18-bp recognition site AGCTTC CCGGGCTCGA GTTAAC-39 was cloned into (8), was used to induce recombination reactions. In previous EcoRI-digested pUC7 DNA. A HindIII cassette containing studies performed with I-Sce I, specific DSBs were induced in the bar gene, which codes for the phosphinothricin acetyltrans- vivo in plasmid DNA transfected or injected into eukaryotic ferase (25), was integrated in the HindIII site. Via PCR, a cells (9–12). Recently I-Sce I-mediated induction of genomic fragment containing the right border and the ‘‘overdrive’’ DSBs and their repair by homologous recombination were sequences of the Agrobacterium strain C58 was cloned between described for mouse cells (13, 14). the Sac I and Xba I sites. Via PCR, a fragment of the The role of genomic DSBs in homologous recombination in kanamycin gene from the plasmid pTZAH271 (26) fused to the plants has not been studied yet. However, several lines of left border of the Agrobacterium strain C58 was obtained and indirect evidence indicate that transient DSBs enhance ho- cloned between the Xma I and Xho I sites of the vector, The publication costs of this article were defrayed in part by page charge Abbreviations: DSB, double-strand break; ORF, open reading frame; payment. This article must therefore be hereby marked ‘‘advertisement’’ in T-DNA, transferred DNA. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be sent at the † address. 5055 5056 Genetics: Puchta et al. Proc. Natl. Acad. Sci. USA 93 (1996) resulting in the plasmid pUC1S. A PCR fragment of the hygromycin gene of pCH was cloned in parallel orientation to the intron sequence (as in pTS) into the Xba I-site of pUC1S. The cloned sequence was excised by digestion with Hpa I and ligated to a Bgl II-digested, blunt-ended, borderless derivative of the binary vector pBin19 (27). The resulting binary vector pRC carried in its transferred DNA (T-DNA) a sequence homologous to both sides of the I-Sce I site of pTS (Fig. 1). All binary plasmids were transferred to the Agrobacterium strain GV3101(pPM6000) (24) and used for plant transformation. Transformation of Tobacco Seedlings with Agrobacterium. Transformations of tobacco seedlings were done as described (28). Three to 4 days after inoculation, the seedlings were transferred onto Murashige and Skoog (MS) plates containing hormones and 100 mg of kanamycin sulfate per ml or 20 mgof glufosinate ammonium per ml. Regeneration of plants from resistant calli was done as described (29). Segregation of the selfed transformants was tested by put- ting seeds onto MS medium supplemented with 50 mgof m hygromycin per ml (target lines), 500 g of kanamycin sulfate FIG. 1. Design of the experiment. pTS represents the target locus, per ml (recombinant lines), or 20 mg of glufosinate ammonium drawn as T-DNA integrated into the plant chromosome. Between the per ml. hygromycin resistance gene (hatched box) and a nonfunctional 39 half Southern Blot Analysis. Total DNA isolation and Southern of the kanamycin resistance gene containing an intron, an I-Sce I site blot analysis were performed as described (30, 31). The probes was cloned. pRC is the repair construct; between the two homologous 9 [kanamycin, HindIII fragment of pAH5 (23) containing the regions, namely, the hygromycin resistance and the 5 end of the complete intron sequence; and hygromycin, Sac II-fragment of kanamycin resistance gene, a phosphinothricin resistance gene (bar) was placed. After homologous recombination induced by a DSB at the pCH (24) containing the complete ORF of hygromycin phos- I-Sce I site, recombinants are expected to have the structure depicted photransferase and parts of the nopaline promoter and ter- in the lower part of the figure. Whereas after Southern blot analysis a 32 minator] were labeled with [ - P]ATP using the random of HindIII (H)-digested DNA, the original target locus should give a primed labeling kit of Boehringer Mannheim. hybridization signal of 3.4 kb, both with a kanamycin- and a hygro- Amplification and Sequence Analysis of the Genomic Re- mycin-specific DNA probe, a recombinant arising by homologous combination Joints. PCR was done as described (29) using the recombination of the left arm of the vector should show a 1.9-kb oligonucleotides pP1 (59-pGCGCAAGCTT CCGACCTGTC kanamycin-specific band, and a recombination of the right arm of the CGGTGCCCTG-39) and pP2 (59-pCCGGAAGCTT vector should show a 1.7-kb hygromycin-specific band. Oligonucleo- CAACGTCGAC CACAGCTGCG-39) (see Fig. 1). The PCR tides P1 and P2 were designed to amplify the recombined intron sequences in the plant genome for sequence analysis. LB, left border; fragment was digested with HindIII and cloned in the RB, right border; 35S, cauliflower mosaic virus 35S promoter; and T, polylinker of pUC18. Sequencing was performed by the Labor cauliflower mosaic virus terminator. fu¨r DNA-Analytik (Freiburg, Germany) using an automated laser florescent sequencing device.
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