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The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence

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The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence (hpvright 0 1997 by the Genetics Society of America The Maize Transposable Element Ac Induces Recombination Between the Donor Site and an Homologous Ectopic Sequence Gil Shalev and Avrzlham A. Levy Department of Plant Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel Manuscript received December 9, 1996 Accepted for publication March 28, 1997 ABSTRACT The prominent repair mechanism of DNA double-strand breaks formed upon excision of the maize Ac transposable element is via nonhomologous end joining. In this work we have studied the role of homologous recombination as an additional repairpathway. To this end, we developed an assay whereby &Glucuronidase (GUS) activity is restored upon recombination between two homologous ectopic (nonal- lelic) sequences in transgenic tobacco plants. One of the recombination partners carried a deletion at the 5’ end of GUS and an Ac or a Ds element inserted at the deletion site. The other partner carried an intact 5’ end of the GUS open reading frame and had a deletion at the 3‘ end of the gene. Based on GUS reactivation data, we found that the excision of Ac induced recombination between ectopic sequences by at least two orders of magnitude. Recombination events, visualized by blue staining, were detected in seedlings, in pollen and in protoplasts. DNA fragments corresponding to recombination events were recovered exclusively in crosses with Ac-carrying plants, providing physical evidence for Ac- induced ectopic recombination.The occurrence of ectopicrecombination following double-strand breaks is a potentially important factor in plant genome evolution. HE transposable element Activator (Ac) of maize cleotide (BANGAand BOYD 1992). Such a gap repair T and the related nonautonomous Dissociation (Ds) mechanism can account for the apparentlack of corre- elements belong to the same class of elements as the lation between excision and reinsertion and forthe En/Spm of maize, P of Drosophila, Tcl of nematodes, effect of an allelic sequence on thereversion frequency TnlO and Tn7 of Escherichia coli, all sharing a conserva- at the donor site. tive “cut-and-paste” mechanism of transposition (see Although in plants HR is not efficient, there are indi- reviews in SAEDLERand GIERL1996). According to this cations that it is induced upon DSB formation: (1) Irra- model, a double-strand break (DSB) is created at the diation was found to enhance the rate of HR (TOVAR donor site upon the element excision. Healing of the and LICHTENSTEIN1992; PUCHTAet al. 1995). (2) Geno- broken donor can occur through a numberof different mic DSB, catalyzed by the yeast I-SceI endonuclease, pathways. In E. coli, where DSB repair via nonhomolo- induces HR between a T-DNA template and a tobacco gous end-joining is not efficient, broken DNA at Tn7 genomic sequence(PUCHTA et al. 1996). (3) An Ac/ or TnlO donor sites is repaired via homologous recom- Ds insertion was shown to increase crossover between bination (HR) (HAGEMANNand CRAIG1993; EICHEN- markers flanking the insertion site (MCCLINTOCK1953; BAUM and LIVNEH 1995).Similarly, HR is involved in GREENBLATT1981). (4) Ac excision from the maize P the donor site repair following P (ENGELSet al. 1990) locus was found to induce recombinationbetween large and Tcl (PLASTERK1991) excision. Based on genetic direct repeats flanking Ac donor site (ATHMAand PE- and molecular characterization of Pand Tcl donor site TERSON 1991). Similarly, the maize Ivfutatorelement was repair, a model was proposed that includes the follow- shown to increase recombination between tandem re- ing successive events: DSB formation upon element ex- peats at the Knl-0 locus (LOUTet al. 1992). cision, gap enlargementvia exonucleolytic degradation The data suggesting that Ac induces HR are in con- exposing 3’ single-strand regions, and invasion of a se- flict withother works showing that an Ac or Ds insertion quence homologous to the donor site to be used as a eitherreduces (MCCLINTOCK1953) or has no effect template for gap repair (ENGELS et al. 1990; PLASTERK (FRADKINand BRINK1956) on crossover in the region 1991; NASSIFet al. 1994). The template for donor site flanking theelement. Moreover, thecorrelation be- repair can be supplied by either the sister chromatid, tween excision and reinsertion of Ac/Ds (GREENBLATT the allelic sequence on the homologue, a nonallelic 1981; CHENet al. 1992) and the lack of apparent allelic effect on the rate of Ds excision suggest that gap repair (ectopic) sequence (ENGELSet al. 1990) or an oligonu- with an homologous template plays a minor role in donor site repair following Ac excision. Finally, the mo- Corrrsponding author: Avrahanl A. Levy, Department of Plant Genet- ics, The Weizmann Institute of Science, Rehovot 76100, Israel. lecular analysis of excision footprints has led to models E-mail: [email protected] where DSBs at the donor sites are repaired by end- Genetics 146 1143-1151 (July, 1997) 1144 Levy G.A. Shalev and A. joining (or illegitimate recombination), following exo- the same sites in the adaptor of pGSOO2 plasmid giving rise nuclease and DNA-polymeraseactivities (COEN et al. to GUS:Ac (pGS004).A Ds-containing plasmid GUS:Ds (pGSOO5) was derived from GUS:Acby filling in the unique 1989; SAEDLERand GIERL1996; SCO~et al. 1996). Ad1site, located inthe codingregion of Ac. In these plasmids To better assess the role of HRin Ac donor site repair, GUS activity can be restored upon element excision. Ac- or we have developed a nonselective assay in transgenic Ds-containing KpnI-SpeI fragments were isolated from tobacco that allowsus to monitor HR in the region pNAOO2, or from pCSOO5, and subcloned into the same sites flanking an Ac or a nonmobile Ds element through in the adaptorof pGSOO3 giving rise to the two recombination partners, 5'AGUS:Ac (pGSOO8) and 5'AGUS:Ds (pGSOO9) , re- blue sectors, which reflect the repair of a defective p- spectively. In these plasmids GUS activity cannot be restored Glucuronidase (GUS) gene. We found that Ac excision upon element excision. induces HR between genomic sequences located at ec- All plasmids described above were subcloned into binary topic (nonallelic) sites by at least two orders of magni- vectors to produce transgenic plants. The HindIII-BglrI frag- tude. In agreement with these findings, recombinant ment of pJD330was subcloned into the same sites of the pGA492 binary vector (AN 1987) giving rise to pGSOO6. The DNA molecules were detected only in Ac- but not in DS HindIII-PVuII fragment containing the 3'AGUS of pGSOOl carrying plants. These data show that in addition to the was subcloned into the HindIII-filled-in CluI sites of the same nonhomologous end-joining repair pathway, HRis used binary vector giving rise to pCSOO7. The 7-kb PstI-BglII GUS:Ac for donor site repair following Ac excision. To our containing fragmentof pGS004 was subcloned into theHpaI- knowledge, this is the first indication of ectopic HR in BamHI sites of the same pGA492 binary vector giving rise to pGS010. The 7-kb PstI-BgZII fragment of 5'AGUS:Ac (pGSOO8) plants. was subcloned into the PstI-BamHI sites of the binary vector pCGN1548 (MCBRIDEand SUMMERFELT1990) giving rise to pGSOl2. Similarly, the blunted 7-kbEcoRI-XbuI fragment of MATERIALSAND METHODS 5'AGUS:Ac (pGS008) was subcloned into the blunted PstI- Plasmid construction: Digestions with restriction enzymes, BglrI sites of the pCPTV-HPT binary vector (BECKERet al. ligations and plasmid preparations were performed according 1992) giving rise to pGS015. The 7-kb PstI-BglII fragments of to standard protocols and manufacturer's instructions (SAM- GUS:Ds (pGS005) and 5'AGUS:Ds (pGSOO9) were ligated with the 14.2-kb PstI-BglII vector-containing fragment of pGS012 BROOK et al. 1989). Plasmid pJD330, kindly provided by V. giving rise to pGSOll and pGSO13, respectively. WALBOT,was used as a positive control forassaying GUS activ- ity. This plasmid is a Bluescript derivative carrying the GUS Tobaccotransformation: The binary vectors mentioned reporter genedriven by the cauliflower mosaic virus 35s pro- above were introduced by electroporation into the Agrobacter- moter fused to the tobacco mosaic virus translationalen- iumtumefuciens LB4404 strain (HOEKEMAet al. 1983). The hancer R (GALLIEet al. 1987). The transcription termination transformed Agrobacterium strains were used to infect leaf fragment of pJD330 is derived from the Nopaline synthase discs of Nicotiana tabacum and regeneratetransgenic plants as (nos) gene ofAgrobactm&m tumefaciens. To test whether Ac described by Horsch et al. (1985). All transgenic plants carried induces HR, we builta series of constructs,derived from transformation markers that confer resistance to kanamycin pJD330, schematically described in Figure 1. Recombination (100 mg/liter) or hygromycin (18 units/ml). Analyses were partners were constructed, each with a different deletion in carried out in progenies of crosses whose parents were hemi- the GUS gene. One partner (3'AGUS), carried a 700 base zygous for one copy of the T-DNA. The number of T-DNA pair (bp) (SspI-EcoRI) deletion in the 3' region of GUS, span- copies was determined on the basis of kanamycin or hygro- ning -500 bp within the GUS gene, and thenos transcription mycin resistance frequency in T2-selfed seedlings and by termination fragment. The 3'AGUS (laboratory number Southern blot analysis (data not shown). pGS001) plasmid was prepared by digestion with EcoRI, blunt- GUS assays: GUS activity in transgenic plants was deter- ing with Klenow, followed by partial Ssp1 digestion and self- mined according to the fluorimetric or histochemical proce- ligation of the 4.5-kilobase (kb) fragment. The second partner dures JEFFERSON et al. 1987). Tissues of transgenicplants consisted of a deletion in the 5' end of GUS with either Ac were stained with the X-Gluc (5-bromo-chloro-3-indolyl-~-D- or Ds inserted at the deletion site. For this purpose pJD330 glucoronidefrom Molecular Probes)substrate and subse- was linearized at the SalI site located between R and the GUS quently cleared as described (BEECKMAN andENCLER 1994).
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