In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type. 1. Introduction Genetic modification of plants is now routinely performed. Transformation can be done by various methods and vectors including Agrobacterium tumefaciens. It has been observed that transgenes integrate at fairly random positions via non-homologous recombination (NHR) in variable copy numbers in the plant genome. This may cause position effects (like silencing of transgenes) and mutation of genes at the integration site. Therefore, it would be an advantage if integration could be targeted to a specific locus. Targeted DNA integration by homologous recombination (HR), also called gene targeting (GT), is efficient in yeast but a very rare event in somatic cells of higher eukaryotes, like animals and plants. DNA repair mutants of the model yeast Saccheromyces cerevisiae were used to identify the genes involved in the integration of the T-DNA. Results showed that genes, which are involved in non-homologous end-joining (NHEJ), are required for integration of the Agrobacterium tumefaciens T-DNA by non-homologous recombination [1]. NHEJ is one of the major DNA repair pathways used to repair double-strand breaks (DSBs). It does not require any homologous sequences and can function throughout the cell cycle [2]. The ends are directly ligated, but this is often accompanied by small deletions and insertions. The other DSB repair pathway is homologous recombination (HR) involving extensive DNA sequence homology between the interacting molecules. HR occurs during
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