Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae.
DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were also sensitive to methylmethane sulfonate (MMS). Ku-dependent repair is distinct from homologous recombination, because deletion of KU80 and HDF1 increased the MMS sensitivity of rad52delta. Interestingly, rad5Odelta, also shown here to be defective in end joining, was epistatic with Ku mutations for MMS repair and end joining. Therefore, Ku and Rad50 participate in an end-joining pathway that is distinct from homologous recombinational repair. Yeast DNA end joining is functionally analogous to DSB repair and V(D)J recombination in mammalian cells.
Milne
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Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Jin
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Shannon
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Weaver
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D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001483:Base Sequence; D004265:DNA Helicases; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D004843:Epistasis, Genetic; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D034961:Antigens, Nuclear; D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C520769:YKU70 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen