Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and nonhomologous end joining

Abstract

Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single double-strand break (DSB) in a plant genome can be mutagenic due to insertions and deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes, each carrying an I-SceI site. After transient expression of I-SceI a kanamycin-resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by nonhomologous end joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB-induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes.

Figure 1.—

Schematic map of the T-DNAs pTS and pTL. Possible outcomes of the recombination reaction are depicted (for reasons of clarity neither promoter nor terminator sequences are shown). HR, homologous recombination; NHEJ, nonhomologous end joining. The triangles represent the primers used for the PCR amplification of the recombined junctions. A 1.2-kb fragment will be detected if the transgene halves A and D are joined by HR. In the case that NHEJ took place, a 2.0-kb fragment is amplified (dashed arrows). The new junction between halves C and B can be detected as a 1.8-kb PCR fragment. E (EcoRV) and H (HindIII) are restriction sites used for Southern blotting (see Figure 3). A 1.9-kb HindIII restricted specific fragment is indicative of HR between transgenes A and D, whereas a 2.7-kb fragment is indicative of NHEJ. The newly joined transgenes C and B are detected as a 4.9-kb fragment in EcoRV-digested genomic DNA with a bar-specific probe. The position and length of the probes used for membrane hybridization are depicted as dashed arrows. RB, right border; LB, left border.

Figure 2.—

Compilation of junctions originating from transgenes B and C in which end joining between the two I-SceI sites had occurred. Assuming that a simple ligation occurred leading to the repair of the two free ends B and C a master sequence was designed that is shown at the top wherein the functional I-SceI site is marked by a shaded frame. Three different classes of junctions are found: in five cases, the I-SceI site was partly destroyed by simple deletions of 2–16 bp. In four recombinant IRC1 lines, deletions of 3–38 bp accompanied the use of microhomologies (underlined letters) at the newly formed junctions of 1–5 bp, a hallmark of DSB repair via NHEJ. A third class of repair events is represented by lines IRC1 2 and IRC1 47 in which insertions (in solid frames) of 61 and 28 bp were detected. These insertions accompanied small deletions of 8 and 1 bp, respectively. Fifty-four nucleotides of the 61-bp insertion of line IRC1 2 (shaded boxes) originate from a part of the transgenic sequence 38 bp downstream of the I-SceI site as depicted above. The sequence is in the same orientation as its original template, which is part of the 3′ end of the 35S terminator. The origins of the last 7 of 61 bp inserted in line IRC1 2 and the 28-bp insertion in line IRC1 47 are unknown.

Figure 3.—

Southern blot analysis with restriction-digested DNA of the plant lines. Lane 1, wild type; lane 2, line 1.12c; lane 3, line 1_12 homo; lane 4, line IRC1; lane 5, line IRC1 7-1; lane 6, line IRC1 12-1; lane 7, line IRC1 12-2; lane 8, line IRC1 25-1; lane 9, line IRC1 52-1. (A) HindIII-restricted DNA hybridized with a kanamycin-intron-specific probe. Wild-type tobacco was used as a negative control (lane 1), whereas lanes 2 and 3 represent positive controls that have been described earlier (see Puchta et al. 1996). Both in the original line 1-12 (which is homozygous for pTS) and in the parental line IRC1 (lane 4) a 3.4-kb kanamycin-intron-specific fragment can be detected. In line IRC1 also a 1.1-kb band can be visualized that corresponds to an HindIII fragment of pTL (see Figure 1). The fragments of the parental line IRC1 are still detectable in the recombinant line IRC1 12-1 (lane 6), and the additional 2.7-kb fragment represents the new joint between transgene halves A and D via NHEJ (see Figure 1). The other lines shown here contain only the recombinant junctions A–D: whereas both lines IRC1 7-1 and 12-2 (lanes 5 and 7, respectively) contain a 2.7-kb fragment, which is representative of NHEJ between transgene halves A and D, for both lines IRC1 25-1 and 52-1 only a 1.9-kb fragment is visible that represents HR-mediated joints between A and D (lanes 8 and 9, respectively). (B) EcoRV-digested DNA hybridized with a bar-specific probe. The controls (lanes 1–3) are the same as those described in A. For the parental line IRC1 (lane 4) a bar-specific 2.4-kb band is detectable, representing an EcoRV fragment of pTL (see Figure 1). This parental band can also be seen in line IRC1 12-1 (lane 6), which also harbors a completely new 4.9-kb band representative of the recombinant junction between transgene halves C and B (see Figure 1). All other recombinant lines shown here (lanes 5 and 7–9) possess only the new 4.9-kb fragment, which is unique for the NHEJ-mediated joint between transgenes C and B.