Different pathways of homologous recombination are used for the repair of double‐strand breaks within tandemly arranged sequences in the plant genome

Abstract

SummaryDifferent DNA repair pathways that use homologous sequences in close proximity to genomic doublestrand breaks (DSBs) result in either an internal deletion or a gene conversion. We determined the ef®ciency of these pathways in somatic plant cells of transgenic Arabidopsis lines by monitoring the restoration of the b-glucuronidase (GUS) marker gene. The transgenes contain a recognition site for the restriction endonuclease I-SceI either between direct GUS repeats to detect deletion formation (DGU.US), or within the GUS gene to detect gene conversion using a nearby donor sequence in direct or inverted orientation (DU.GUS and IU.GUS). Without expression of I-SceI, the frequency of homologous recombination (HR) was low and similar for all three constructs. By crossing the different lines with an I-SceI expressing line, DSB repair was induced, and resulted in one to two orders of magnitude higher recombination frequency. The frequencies obtained with the DGU.US construct were about ®ve times higher than those obtained with DU.GUS and IU.GUS, irrespective of the orientation of the donor sequence. Our results indicate that recombination associated with deletions is the most ef®cient pathway of homologous DSB repair in plants. However, DSB-induced gene conversion seems to be frequent enough to play a signi®cant role in the evolution of tandemly arranged gene families like resistance genes.

In planta gene targeting

“…GUS assays were performed as described (39). Determination of GT events that entered the germline was performed with 10-d-old seedlings (F2′ generation), cultured on sand as described (40).…”

Repair of Site-Specific DNA Double-Strand Breaks in Barley Occurs via Diverse Pathways Primarily Involving the Sister Chromatid

“…Although all three constructs showed a deletion bias (on average 6.5 classes with a net deletion per one repair class with a net insertion), within the frame of resolution of 900 to 1400 bp, respectively, by far the most deletions were smaller than 100 bp, and thus clearly below the size of deletions reported for Arabidopsis calluses (Kirik et al, 2000). The lack of reads with a 652-bp deletion within the GU.US library 12A that would have resulted in a functional GUS gene by SSA (which is 5 times more efficient than functional GUS formation via SDSA in Arabidopsis; Orel et al 2003) also supports the infrequent occurrence of large deletions during DSB repair in barley. Thus, our results are concordant with the hypothesis that very small genomes are the result of a stronger bias toward long deletions during DSB repair (Kirik et al, 2000;Puchta, 2005).…”

“…This observation suggests that, in contrast to the presumed predominant intrachromatid repair in Arabidopsis (Orel et al, 2003) and in tobacco (Siebert and Puchta, 2002), barley uses the sister chromatid in the majority of cases for repair of staggered DSBs, apparently independent of whether immediate ligation, SDSA, NHEJ, or MMHJ is followed to seal the break. A few hints that SCE could be a major pathway of DSB repair came from the observation of increased SCE frequency after treatment of mammalian cells with restriction endonucleases (Natarajan et al, 1985) and from Rad51 (recombinase)-dependent equal SCE in broken centric monoplasmids of yeast (González-Barrera et al, 2003).…”

“…Alternatively, repair-mediated loss of a transgenic marker gene (negative selection) has been used to interpret repair processes that did not restore the original sequence context. Orel et al (2003) showed that in Arabidopsis, SSA restored a functional gene about 5 times more efficiently than SDSA and that the template used for gene conversion by SDSA was independent of its orientation toward the target site (DU.GUS versus IU.GUS). Blue spots indicating GUS activity appeared 1 to 2 orders of magnitude above the spontaneous frequency after transient DSB induction.…”

“…Because no system was investigated comprehensively for all potential mechanisms, we have only insufficient knowledge about the quantitative contribution of the individual options for DSB repair. Therefore, we aimed to estimate the proportions of different DSB repair pathways at the molecular and chromosomal level by tracing the fate of DSBs targeted to three b-glucuronidase reporter transgene variants (GU.US, IU.GUS, and DU.GUS; Orel et al, 2003) in the monocotyledoneous plant barley (Hordeum vulgare). The reporter genes are interrupted by 38 bp in GU.US and 30 bp in DU.GUS and IU.GUS.…”