| Double-strand break (DSB) repair pathways catalyze the rejoining of broken chromosomes and the integration of transforming DNAs. Plants are thought to use primarily a non-homologous end-joining (NHEJ) pathway to repair DSBs and integrate transgenes. We isolated T-DNA insertional mutations defective in the Arabidopsis homologs of the Ku80 and DNA Ligase IV (LIG4) genes, known from mammalian and yeast studies to be required for the initiation and completion, respectively, of NHEJ. Both mutants are indistinguishable from their wild-type progenitors in the absence of DNA-damaging agents, but hypersensitive to the cytostatic effects of ionizing radiation (IR) suggesting that NHEJ is a critical pathway for DSB repair in plants as in mammals. We also find that the homozygous mutants exhibit decreased levels of random T-DNA integration; lig4 is transformed at about 40--70%, and ku80 at about 15--40%, of wild-type levels. These data provide support for the involvement of AtKu80 and AtLIG4 in random integration although they are not essential for the process. In order to determine whether homology-dependent T-DNA integration is enriched in ku80, lig4 double mutants we attempted gene replacement of an endogenous plant locus. Our data suggest that gene replacement is not highly enriched in the absence of Ku80 and LIG4.; The histone variant H2AX is rapidly and massively phosphorylated at the sites of DSBs. This phosphorylated H2AX (gamma-H2AX) is involved in the retention of repair and signaling factor complexes at sites of DNA damage. The dependency of this phosphorylation on the various PIK3-related protein kinases (in mammals, ATR, ATM, and DNA-PKcs) has been a subject of debate. Using Arabidopsis as a model system, we investigated the ATR- and ATM-dependency of the formation of gamma-H2AX foci in M phase cells exposed to IR. We find that while the majority of these foci are ATM-dependent, approximately 10% of IR-induced gamma-H2AX foci require, instead, functional ATR. This indicates that even in the absence of DNA replication, a distinct subset of IR induced damage is recognized by ATR. In addition, we find that in plants, gamma-H2AX foci are induced at only 1/4th the rate observed in yeasts and mammals. |
