Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Abstract

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 ofthe 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. (7), which has an 18-bp recognition site (8), was used to induce recombination reactions. In previous studies performed with I-Sce I, specific DSBs were induced in vivo in plasmid DNA transfected or injected into eukaryotic cells (9-12). Recently I-Sce I-mediated induction of genomic DSBs and their repair by homologous recombination were described for mouse cells (13,14). We demonstrated that expression of the I-Sce I gene led to specific DSBs in vivo in DNA molecules transfected into plant protoplasts (10). As a consequence extrachromosomal homologous recombination was induced. This reaction proceeds mainly via single-strand annealing and thus seems to be different from recombination reactions in the chromosome (as discussed in ref. 20). In the present communication we analyzed the mechanism by which DSBs at specific sites in the plant genome are repaired with homologous sequence information, and we tested whether induction of DSBs is correlated with an increase in recombination frequencies. MATERIALS AND METHODSCloning Procedures. Construction of the I-Sce I expression 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 cut with BamHI and the ORF containing fragment was inserted in the unique BamHI site of the binary vector pCGN1589 (21) to result in the plasmid pCISceI.Construction of the target site. Into the EcoRV site of the plasmid pAF100 (22), the Xho I-Acc65I-cut, blunt-ended DNA fragment of the plasmid pAH5 (23) carrying the C-terminal half of an artificial, intron-containing kanamycin resistance gene was cloned....

The plant‐specific CDKB 1‐ CYCB 1 complex mediates homologous recombination repair in Arabidopsis

“…Previous experiments suggested that NHEJ and not HR is the predominant type of DNA repair in plants, a situation reminiscent to mammals but different to yeast in which HR is preferred over NHEJ (Puchta et al , 1996; Kempin et al , 1997; Gisler et al , 2002). However, our work clearly indicates that HR is executed under DNA damage conditions in plants and depends on the presence of CDKB1s and CYCB1s.…”

Abortive Gap Repair: Underlying Mechanism for Ds element Formation

“…6C). Such one-sided invasion is reminiscent of the onesided invasion pathway, which has been proposed as a mechanism for DSB repair in mammals (2) and in plants (47), with one important difference; i.e., while in SDSA, the bubble migration releases single-strand DNA, whereas with one-sided invasion, a large and stable heteroduplex is formed.…”

High-frequency gene targeting in Arabidopsis plants expressing the yeast RAD54 gene

“…It has been reported that gene-targeting efficiency can be increased in plants by using I-SceI, a site-specific endonuclease that mediates DNA double-strand breaks, and its recognition site, which creates a recombination hotspot in the chromosome (11). However, this approach requires the prior insertion of an I-SceI recognition site into the genome.…”