Abstract
DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1–20 bp) deletions occurred at a minority (25–45%) of repair junctions. Approximately ∼1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.
Highlights
DNA double strand breaks (DSBs) that occur frequently in eukaryotes are potentially lethal to the cell as they lead to mitotically unstable acentric chromosome fragments and the consequent loss of essential genes [1]
Following DSB induction the plants were left for several days to allow DSB repair to take place, after which the tissue was harvested and DNA prepared
Template molecules which contain the spacer region may have been repaired through homologous recombination using the sister chromatid as a template or, alternatively, these molecules may come from cells in which DSBs were not induced
Summary
DNA double strand breaks (DSBs) that occur frequently in eukaryotes are potentially lethal to the cell as they lead to mitotically unstable acentric chromosome fragments and the consequent loss of essential genes [1]. DNA DSBs may be repaired without the use of a homologous template by using nonhomologous end joining (NHEJ) [1]. In plants this latter pathway appears responsible for the majority of DSB repair [4]. AltNHEJ, sometimes referred to as backup-NHEJ (B-NHEJ) [7] or micro-homology-mediated end joining (MMEJ) [8], is not as well characterised and may well include several distinct repair pathways [1]. NHEJ promotes cancer cell survival [11] and inhibitors of NHEJ can be used to increase the sensitivity of tumours to DNA damaging drugs [12] or radiation treatment [13]
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