Defining the minimal domain of Ku80 for interaction with Ku70.
The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.
Osipovich
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Laboratory of Molecular Immunoregulation, NCI, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Durum
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Muegge
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D034961:Antigens, Nuclear; D001665:Binding Sites; D019976:Cloning, Organism; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C081647:high affinity DNA-binding factor, S cerevisiae; D001616:beta-Galactosidase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen