Disruption of the Arabidopsis RAD50 gene leads to plant sterility and MMS sensitivity

Abstract

The Rad50 protein is involved in the cellular response to DNA-double strand breaks (DSBs), including the detection of damage, activation of cell-cycle checkpoints, and DSB repair via recombination. It is essential for meiosis in yeast, is involved in telomere maintenance, and is essential for cellular viability in mice. Here we present the isolation, sequence and characterization of the Arabidopsis thaliana RAD50 homologue (AtRAD50) and an Arabidopsis mutant of this gene. A single copy of this gene is present in the Arabidopsis genome, located on chromosome II. Northern analysis shows a single 4.3 Kb mRNA species in all plant tissues tested, which is strongly enriched in flowers and other tissues with many dividing cells. The predicted protein presents strong conservation with the other known Rad50 homologues of the amino- and carboxy-terminal regions. Mutant plants present a sterility phenotype which co-segregates with the T-DNA insertion. Molecular analysis of the mutant plants shows that the sterility phenotype is present only in the plants homozygous for the T-DNA insertion. An in vitro mutant cell line, derived from the mutant plant, shows a clear hypersensitivity to the DNA-damaging agent methylmethane sulphonate, suggesting a role of RAD50 in double-strand break repair in plant cells. This is the first report of a plant mutated in a protein of the Rad50-Mre11-Xrs2 complex, as well as the first data suggesting the involvement of the Rad50 homologue protein in meiosis and DNA repair in plants.

Double-stranded DNA breaks and gene functions in recombination and meiosis

“…Abnormalities in mitotic development is not obvious under normal growth conditions; however, mutant plants exhibit an increased sensitivity to the DNA-damaging agent methylmethane sulfonate [57]. An Arabidopsis atmre11 mutant meiocytes lack chromosome synapsis, and both mitotic and meiotic mutant cells contain AtSPO11-1-dependent chromosome fragments, suggesting that this gene functions downstream of AtSPO11-1; this is consistent with the function of RAD50-MRE11-XRS2 complex in budding yeast [35].…”

“…In the Arabidopsis atrad50 mutant, chromosomes fail to synapse and the processing of meiotic DSBs is defective, resulting in the fragmentation of meiotic chromosomes that are visible starting at premetaphase I [56,57]. Abnormalities in mitotic development is not obvious under normal growth conditions; however, mutant plants exhibit an increased sensitivity to the DNA-damaging agent methylmethane sulfonate [57].…”

Inactivation of Mre11 Does Not Affect VSG Gene Duplication Mediated by Homologous Recombination in Trypanosoma brucei

“…Usually, inactivation of Mre11 leads to increased sensitivity to MMS (3,7,20,22,53), although this is not always the case when the NBS1 component of the human M/R complex is mutated (54). Nonetheless, the sensitivity of the trypanosome null mutant to phleomycin and its altered integration patterns do show that Mre11 is involved in repair and recombination.…”

The multiple roles of the Mre11 complex for meiotic recombination

“…In particular, all organisms tested show defects in meiotic recombination in Mre11 complex mutants, e.g. in C. elegans (Chin & Villeneuve 2001), in Coprinus cinereus (Ramesh & Zolan 1995, Gerecke & Zolan 2000, Merino et al 2000, in Arabidopsis thaliana (Gallego et al 2001, Bundock & Hooykaas 2002, Puizina et al 2004, in Aspergillus nidulans (Semighini et al 2003, Malavazi et al 2005 or in Schizzosaccharomyces pombe (Young et al 2004).…”

“…In the plant Arabidopsis thaliana a truncated mutant of Rad50, lacking the last 266 amino acids, is hypersensitive to MMS and sterile (Gallego et al 2001). Similarly, a Mre11 mutant (the mre11-1 allele, truncated after amino acid 261, in the phosphoesterase motif IV) is viable, but sterile (Bundock & Hooykaas 2002).…”