Dataset Information

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining.

ABSTRACT: To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70-/- and AtLig4-/-). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70-/- mutant was slightly more sensitive than the AtLig4-/- mutant. Single-base substitutions (SBSs) were the predominant mutations in the wild-type control, whereas deletions (?2 bp) and complex-type mutations [i.e. more than two SBSs or short insertion and deletions (InDels) separated by fewer than 10 bp] were frequently induced in the mutants. Single-base deletions were the most frequent deletions in the wild-type control, whereas the most common deletions in the mutants were 11-30 bp. The apparent microhomology at the rejoined sites of deletions peaked at 2 bp in the wild-type control, but was 3-4 bp in the mutants. This suggests the involvement of alternative end joining and single-strand annealing pathways involving increased microhomology for rejoining DNA ends. Complex-type mutations comprising short InDels were frequently detected in the mutants, but not in the wild-type control. Accordingly, NHEJ is more precise than the backup pathways, and is the main pathway for rejoining the broken DNA ends induced by ionizing radiation in plants.

SUBMITTER: Du Y

PROVIDER: S-EPMC7482170 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining.

Du Yan Y Hase Yoshihiro Y Satoh Katsuya K Shikazono Naoya N

Journal of radiation research 20200901 5

To investigate the involvement of the non-homologous end joining (NHEJ) pathway in plant mutagenesis by ionizing radiation, we conducted a genome-wide characterization of the mutations induced by gamma rays in NHEJ-deficient Arabidopsis mutants (AtKu70-/- and AtLig4-/-). Although both mutants were more sensitive to gamma rays than the wild-type control, the AtKu70-/- mutant was slightly more sensitive than the AtLig4-/- mutant. Single-base substitutions (SBSs) were the predominant mutations in t ...[more]

PMID: 32766789

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Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Experiment Overall Design: Number of plants pooled:20

Project description:The proposal aims to characterise the pathways of DSB repair and recombination in Arabidopsis with the main focus of this research being the NHEJ pathway of illegitimate recombination. We will build on our expertise and resources in the field of DSB repair in plants, using the Arabidopsis NHEJ mutant atku80 which is an excellent model system for the study of DSB repair in higher eukaryotes (West et al., 2002 Plant J. 31, 517-28). Comparison of the transcriptome in NHEJ mutant and wild type plants under different experimental conditions will identify novel candidate genes involved in DNA DSB repair or damage signalling pathways.We will conduct 4 separate experiments on Arabidopsis seedlings grown on 0.5 MS media: the first will be a control consisting of Wassilewskija (WS-2) plants grown under standard conditions. In the second experiment WS plants will be exposed to the chemotherapeutic agent bleomycin (1 microgram per ml). This agent has been shown to cause both single and double strand breaks in the genome of Arabidopsis seedlings (Menke et al., 2001 Mut. Res. 493, 87-93). This agent is used in preference to gamma irradiation, which causes high levels of oxidative damage to cellular components. These experiments will characterise for the first time the transcriptional responses of Arabidopsis cells to the specific induction of DNA strand breaks. The transcript profile will also be determined in untreated atku80 mutants. This experiment will identify the components of novel and previously characterised DNA repair pathways up regulated in the mutant background. Finally, transcript analysis of bleomycin treated atku80 mutants and comparison with untreated mutant plants and both untreated and treated WS plants will identify novel putative DSB repair genes up regulated in response to genotoxic stress in the absence of a Ku80 mediated DSB repair pathway.The results of these studies will provide new and important information which will be instrumental in identifying novel genes and pathways involved in DNA repair and genome stability in plants and help elucidate the fundamental differences that exist between animal and plant DSB repair processes. Keywords: strain_or_line_design; compound_treatment_design

Dataset Information

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining.

Publications

Characterization of gamma irradiation-induced mutations in Arabidopsis mutants deficient in non-homologous end joining.

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