Double-Strand Break Repair in Plants Is Developmentally Regulated<sup><a href="#fn1" rid="fn1" class=" fn">1</a>,</sup><sup><a href="#fn2" rid="fn2" class=" fn">[W]</a></sup>
Abstract
In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional β-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 × 10) being only 30% of the recombination rate at 2 dpg (9.14 × 10). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.
The genetic material of any organism is constantly fluctuating, with hundreds of mutations varying from silent-base substitutions to large deletions/insertions being introduced upon each genome replication (Tuteja et al., 2001; Kunz et al., 2005). The frequency with which these mistakes persist depends on several parameters, such as the competence of polymerase proofreading activity, the effectiveness of the proteins involved in early DNA damage recognition, the efficiency of chromatin modifiers, and the precision of the core DNA repair enzymes. In many cases, the same type of lesion can be repaired by several different DNA surveillance mechanisms. The balance between these mechanisms maintains the relative genome stability of a given organism.
Single- and double-strand breaks (SSBs and DSBs) are good examples of the lesions that are processed by the various repair pathways broadly grouped to nonhomologous end joining (NHEJ) and homologous recombination (HR; Sargent et al., 1997; Liang et al., 1998). These lesions can be extremely deleterious as even a single, unprocessed break may lead to cell death (Karanjawala et al., 2002).
NHEJ and HR have different repair fidelities. NHEJ is believed to result in various mutations, varying from single nucleotide substitutions to deletions/insertions of one to several thousand nucleotides (Roth and Wilson, 1986; Brennan and Schiestl, 1998; Jeggo, 1998; Ries et al., 2000; Ikeda et al., 2001; Pelczar et al., 2003; Kovalchuk et al., 2004). Conversely, HR, although generally believed to be free of repair mistakes, frequently results in large segmental duplications, gene duplication, gene loss, or gene inactivation. Presently, it is not clear which mechanism more significantly contributes to genome rearrangements and, therefore, to genome evolution (Gorbunova and Levy, 1997; Critchlow and Jackson, 1998; Kirik et al., 2000; Smith et al., 2001).
It has been well documented that the contribution of either NHEJ or HR to the repair of strand breaks varies from organism to organism (Cromie et al., 2001) and from tissue to tissue (Essers et al., 2000). For example, the frequency of HR-based repair was found to be different in various tissues of mammalian organisms, whereby embryonic stem cells displayed a higher frequency of HR when compared to other, differentiated cells (Essers et al., 2000). Moreover, various areas of the genome appear to have different rates of HR (Puchta et al., 1995; Filkowski et al., 2004) and, perhaps, NHEJ (Kovalchuk et al., 2000). However, the phenomenon of cell type-specific HR rates, as displayed in mammals, to the best of our knowledge, has not been studied in plants.
Given that the rates of HR and NHEJ differ according to the circumstance, the contribution of various DNA repair mechanisms to each specific lesion may also vary at different stages of organism development. Providing that the efficiency of any process depends on two major factors, cost and precision, the balance between reasonable costs and reasonable precision defines what is typical organism development. Therefore, as HR and NHEJ have different repair fidelities and different costs, their contribution to strand break repair at different developmental stages may also vary.
In this study we analyzed the HR events in Arabidopsis (Arabidopsis thaliana) at different developmental stages. We found that the rate of HR decreased with plant age, while the frequency of strand breaks and point mutation rates increased. These results were paralleled by a decrease in the abundance of Rad51 and an increase in the abundance of Ku70 transcripts. This phenomenon may reflect the existence of a mechanism that provides tight control over extensive recombination in polyploid plant cells.
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We would like to thank Chris Picken for critical reading of the manuscript. We acknowledge the Natural Sciences and Engineering Research Council of Canada and Alberta Heritage for Science and Engineering grants for I.K.
Notes
This work was supported by the Natural Sciences and Engineering Research Council of Canada (Establishment Grant to I.K.).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Igor Kovalchuk (ac.htelu@kuhclavok.rogi).
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Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.074658.