Managing Editor
Marisa Rosa
  • Managing Editor, Bio-protocol
Research fields
  • Plant Science
Measuring Homologous Recombination Frequency in Arabidopsis Seedlings
Authors:  Marisa Rosa and Ortrun Mittelsten Scheid, date: 04/05/2014, view: 12186, Q&A: 0
Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to increase in response to various chemical and physical agents that cause DNA damage and/or alter genome stability (reviewed in March-Díaz and Reyes, 2009). We monitor the frequency of SHR in transgenic Arabidopsis seedlings containing recombination substrates with two truncated but overlapping parts of the β-glucuronidase (GUS) reporter gene (Orel et al., 2003; Schuermann et al., 2005). Upon an SHR event, a functional version of the transgene can be restored (Figure 1A). A histochemical assay applicable to whole plantlets allows the visualization of cells in which the reporter is restored, as the encoded enzyme converts a colorless substrate into a blue compound. This type of reporter has been extensively used to identify gene products required for regulating SHR levels in plants. We analyze plants stimulated for SHR by treatments with DNA damaging agents (bleocin, mitomycin C and UV-C) and compare them to non-treated plants.
DNA Damage Sensitivity Assays with Arabidopsis Seedlings
Authors:  Marisa Rosa and Ortrun Mittelsten Scheid, date: 04/05/2014, view: 10479, Q&A: 0
We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected.
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