title: Molecular and biochemical characterization of xrs mutants defective in Ku80.
abstract: The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.
authors.affiliation: MRC Cell Mutation Unit, University of Sussex, Brighton, United Kingdom.
authors.forename: B K
authors.initials: BK
authors.lastname: Singleton
authors.forename: A
authors.initials: A
authors.lastname: Priestley
authors.forename: H
authors.initials: H
authors.lastname: Steingrimsdottir
authors.forename: D
authors.initials: D
authors.lastname: Gell
authors.forename: T
authors.initials: T
authors.lastname: Blunt
authors.forename: S P
authors.initials: SP
authors.lastname: Jackson
authors.forename: A R
authors.initials: AR
authors.lastname: Lehmann
authors.forename: P A
authors.initials: PA
authors.lastname: Jeggo
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear; D001374:Azacitidine / Q000494:pharmacology; D016466:CHO Cells / Q000528:radiation effects; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases; D018076:DNA, Complementary / Q000235:genetics; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005720:Gamma Rays; D018628:Gene Dosage; D005816:Genetic Complementation Test; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000032:analysis; D011836:Radiation Tolerance / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D001374:Azacitidine