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Type: Journal article
Title: Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and arabidopsis
Author: Lloyd, A.
Wang, D.
Timmis, J.
Citation: PLoS One, 2012; 7(2):1-10
Publisher: Public Library of Science
Issue Date: 2012
ISSN: 1932-6203
Statement of
Andrew H. Lloyd, Dong Wang and Jeremy N. Timmis
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 nonclassical 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.
Keywords: Arabidopsis; Tobacco; DNA, Plant; Polymerase Chain Reaction; DNA Repair; DNA Breaks, Double-Stranded
Rights: Copyright: © 2012 Lloyd et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RMID: 0020117418
DOI: 10.1371/journal.pone.0032255
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Appears in Collections:Molecular and Biomedical Science publications

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