MUTATIONS IN 2 KU HOMOLOGS DEFINE A DNA END-JOINING REPAIR PATHWAY INSACCHAROMYCES-CEREVISIAE

DNA double-strand break (DSB) repair in mammalian cells is dependent o n the Ku DNA binding protein complex. However, the mechanism of Ku-med iated repair is not understood. We discovered a Saccharomyces cerevisi ae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku80 associates with the product of the HDFI gene, forming t he major DNA end-binding complex of yeast cells. DNA end binding was a bsent in ku80 Delta, hdfl Delta, or ku80 Delta hdfl Delta strains. Ant isera specific for epitope tags on Ku80 and Hdfl were used in supershi ft and immunodepletion experiments to show that both proteins are dire ctly involved in DNA end binding. In vivo, the efficiency of two DNA e nd-joining processes were reduced >10-fold in ku80 Delta, hdf7 Delta, or ku80 Delta hdfl Delta strains: repair of linear plasmid DNA and rep air of an HO endonuclease-induced chromesomal DSB. These DNA-joining d efects correlated with DNA damage sensitivity, because ku80 Delta and hdfl Delta strains were also sensitive to methylmethane sulfonate (MMS ). Ku-dependent repair is distinct from homologous recombination, beca use deletion of KU80 and HDFI increased the MMS sensitivity of rad52 D elta Interestingly, rad5o Delta, also shown here to be defective in en d joining, was epistatic with Ku mutations for MMS repair and end join ing. Therefore, Ku and Rad50 participate in an end-joining pathway tha t is distinct from homologous recombinational repair. Yeast DNA end jo ining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.