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Molecular and biochemical characterization of xrs mutants defective in Ku80

Citation

Singleton, BK and Priestley, A and Steingrimsdottir, H and Gell, D and Blunt, T and Jackson, SP and Lehmann, AR and Jeggo, PA, Molecular and biochemical characterization of xrs mutants defective in Ku80, Molecular and Cellular Biology, 17, (3) pp. 1264-1273. ISSN 0270-7306 (1997) [Refereed Article]

DOI: doi:10.1128/MCB.17.3.1264

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.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Biochemistry and Cell Biology
Research Field:Enzymes
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Biological Sciences
Author:Gell, D (Dr David Gell)
ID Code:64591
Year Published:1997
Web of Science® Times Cited:151
Deposited By:Menzies Institute for Medical Research
Deposited On:2010-08-12
Last Modified:2011-08-15
Downloads:0

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