Measuring Homologous Recombination Frequency in Arabidopsis Seedlings

Abstract

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.

Figure 1. Principle of the assay.

(A) Recombination substrates in reporter lines for SHR DGU.US-1 and IU.GUS-8 (adapted from Reference 2). These lines were designed to distinguish different somatic recombination pathways (single-strand annealing and synthesis-dependent strand annealing) upon DSB repair after expression of the endonuclease I-SceI (Orel et al., 2003; for more information about different SHR pathways see Reference 1). However, they are used here to measure SHR without I-SceI but after different exogenous DNA damage treatments. The DGU.US-1 substrate contains two fragments of the GUS ORF with homologous parts (GU, US) in direct orientation, harboring an overlap of 557 bp. In IU.GUS-8, two fragments of the GUS ORF (“GU”, “US”; blue) in direct orientation are separated, rendering the gene not functional. A fragment of 1087 bp partially homologous to “GU” and “US”, and containing “U” is located upstream in inverted orientation. SHR events between the repeats can restore a functional gene (GUS) in different ways: in line DGU.GUS-1, the sequence in common between the two repeats is deleted, while HR between indirect repeats in IU.GUS-8 results in the restoration of the GUS gene. P: constitutive 35S promoter of cauliflower mosaic virus; T: nopaline synthase terminator; HPT: hygromycin resistance gene, BAR: phosphinothricin acetyltransferase resistance gene, GUS: β -glucuronidase gene. (B) Histochemical GUS staining allows to visualize cells in which the GUS gene was restored by SHR (from Orel et al., 2003).

Figure 2. Examples of SHR frequency measurements.

Distribution of seedlings with different numbers of blue sectors and total frequency in line IU.GUS-8. A Mock and B 1 μg/ml Bleocin treatment. Sectors/plant refers to the average number of blue sectors, corresponding to recombination events, in the analyzed seedling population. Error bars correspond to the SE. SHR frequencies in each mutant population were significantly different to their wild-type (WT) counterpart, with a P value < 0.001. Asterisks indicate the significance between treated and mock populations according to P values from unpaired t tests: ***P < 0.001 and **0.001 < P < 0.01.