Dataset Information

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

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.

SUBMITTER: Lloyd AH

PROVIDER: S-EPMC3289645 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

Lloyd Andrew H AH Wang Dong D Timmis Jeremy N JN

PloS one 20120228 2

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 N ...[more]

PMID: 22389691

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Project description:Double strand-breaks (DSBs) of genomic DNA caused by ionizing radiation or mutagenic chemicals are a common source of mutation, recombination, chromosomal aberration, and cell death. Linker histones are DNA packaging proteins with established roles in chromatin compaction, gene transcription, and in homologous recombination (HR)-mediated DNA repair. Using a machine-learning model for functional prioritization of eukaryotic post-translational modifications (PTMs) in combination with genetic and biochemical experiments with the yeast linker histone, Hho1, we discovered that site-specific phosphorylation sites regulate HR and HR-mediated DSB repair. Five total sites were investigated (T10, S65, S141, S173, and S174), ranging from high to low function potential as determined by the model. Of these, we confirmed S173/174 are phosphorylated in yeast by mass spectrometry and found no evidence of phosphorylation at the other sites. Phospho-nullifying mutations at these two sites results in a significant decrease in HR-mediated DSB repair templated either with oligonucleotides or a homologous chromosome, while phospho-mimicing mutations have no effect. S65, corresponding to a mammalian phosphosite that is conserved in yeast, exhibited similar effects. None of the mutations affected base- or nucleotide-excision repair, nor did they disrupt non-homologous end joining or RNA-mediated repair of DSBs when sequence heterology between the break and repair template strands was low. More extensive analysis of the S174 phospho-null mutant revealed that its repression of HR and DSB repair is proportional to the degree of sequence heterology between DSB ends and the HR repair template. Taken together, these data demonstrate the utility of machine learning for the discovery of functional PTM hotspots, reveal linker histone phosphorylation sites necessary for HR and HR-mediated DSB repair, and provide insight into the context-dependent control of DNA integrity by the yeast linker histone Hho1.

Dataset Information

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

Publications

Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

Similar Datasets

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