Frozen leaf cells was pulverized with a mortar and pestle in liquid nitrogen, and total genomic DNA was isolated as described previously (Hong et al

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Frozen leaf cells was pulverized with a mortar and pestle in liquid nitrogen, and total genomic DNA was isolated as described previously (Hong et al., 2005). restriction fragment analysis revealed that telomeres in LY-900009 homozygous G2osku70mutants were markedly longer (1020 kb) than those in wild-type plants (510 kb), whereas telomere length in heterozygous G2osku70mutant and T2OsKu70-overexpressing transgenic (35S:OsKu70) rice resembled that of the wild-type herb. In contrast to what was observed in Arabidopsis (Arabidopsis thaliana)atku70mutants, homozygous G2osku70rice plants displayed severe developmental defects in both vegetative and reproductive organs under normal growth conditions, resulting in sterile flowers. Analysis of meiotic progression in pollen mother cells exhibited that up to 11.1% (seven of 63) of G2 mutant anaphase cells displayed one or more chromosomal fusions. These results suggest that OsKu70 is required for the maintenance of chromosome stability and normal developmental growth in rice plants. Higher plants are constantly exposed to various environmental stresses, including ionizing radiation and genotoxic brokers. These stresses cause DNA damage, including double-strand breaks (DSBs). The repair of DSBs is essential for maintaining the integrity and function of the genome. The major DSB repair mechanism in prokaryotes is usually homologous recombination, whereas nonhomologous end joining (NHEJ) is usually predominant over homologous recombination in higher eukaryotes during G1 phase (Lieber and Karanjawala, 2004). The NHEJ pathway is usually modulated by the DNA-dependent protein kinase (DNA-PK) LY-900009 complex, which consists of a Ku70/Ku80 heterodimer and a catalytic subunit (DNA-PKcs), and the DNA ligase IV (Lig4)/Xrcc4 (Lif1) complex in vertebrates (Critchlow and Jackson, 1998). The first step in the NHEJ pathway is usually recognition of DSB LY-900009 termini and binding of uncovered DNA ends by Ku70/Ku80 heterodimers, which recruit DNA-PKcs and activate its kinase activity (Dynan and Yoo, 1998). The Ku and DNA-PKcs complexes are localized exclusively to the nucleus and have strong affinity for double-stranded DNA ends, impartial of DNA sequence and structure (Koike et al., 1998;Wang et al., 1998). In addition, they participate in RNA polymerase I- and II-mediated transcription, in DNA replication, and in regulation of cell cycle progression (Tuteja and Tuteja, 2000). In yeast cells, a Ku70/Ku80 heterodimer is required not only for DNA repair but also for the maintenance of telomere length. The Ku complex binds to telomeric repeats in vivo, and Ku70-deficient cells exhibit shortened telomeres (Boulton and Jackson, 1998;Baumann and Cech, 2000). In human, Ku interacts with hTERT and the telomerase RNA subunit, perhaps to regulate access of telomerase to telomeric DNA ends (Chai et al., Rabbit Polyclonal to DYR1B 2002;Stellwagen et al., 2003). Ku70 appears to prevent end-to-end chromosome fusion and to bind the telomere repeat-binding protein TRF2 (Hsu et al., 2000;Track et al., 2000). It was recently exhibited that Ku86 is essential for human telomere integrity and that its inactivation leads to cell death along with massive loss of telomeric DNA via recombination in human somatic cells (Wang et al., 2009). Although the DNA repair mechanisms in higher plants are less well comprehended than those in yeast or mammals, components involved in plant DSB repair have been identified and some aspects of their mechanisms have been worked out (Gorbunova and Levy, 1999;Gallego and White, 2005;Riha et al., 2006). For instance, Arabidopsis (Arabidopsis thaliana) AtKu70/AtKu80 heterodimers possess single-stranded DNA-dependent ATPase and ATP-dependent DNA helicase activities in vitro (Tamura et al., 2002). Moreover, theAtKu70andAtKu80genes are significantly up-regulated by DNA-damaging drugs in cultured Arabidopsis cells. These findings raise the possibility that AtKu70 and AtKu80 are involved in DSB repair through the NHEJ system (Tamura et al., 2002). Indeed, althoughAtKu70T-DNA knockout plants appeared normal under standard growth conditions, their seedlings were highly sensitive to -irradiation that induced DSBs (Bundock et al., 2002;Riha et al., 2002). Similarly, disruption ofAtKu80resulted in hypersensitivity to genotoxic brokers (West et al., 2002;Gallego et al., 2003a), consistent with the notion that AtKu70/AtKu80 proteins play a role in DSB repair in Arabidopsis. In contrast to what was observed in yeast and some mammalian cells, mutations inAtKu70andAtKu80resulted in longer telomeres in Arabidopsis, suggesting their role in telomere regulation as negative factors (Bundock et al., 2002;Riha et al., 2002;Gallego et al., 2003b;Riha and Shippen, 2003;Watson and Shippen, 2007). Interestingly, in Arabidopsis, critically shortened telomeres (<300400 bp).