MOLECULAR AND BIOCHEMICAL-CHARACTERIZATION OF XRS MUTANTS DEFECTIVE IN KU80

The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensi vely studied xrs mutants, has recently been identified as Ku80, a subu nit 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 exa mined 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, h as Ku80 transcript levels detectable by Northern hybridization, but Ku 80 mRNA was detectable by reverse transcription-PCR in most other muta nts. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resultin g from mutational changes in the genomic Ku80 sequence affecting RNA s plicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these t wo mutants has detectable wild-type Ku80 transcript. Since the mutatio n in both xrs-4 and xrs-6 cells results in severely truncated Ku80 pro tein, both are likely candidates to he null mutants. Azacytidine-induc ed revertants of xrs-4 and -6 carried both wild-type and mutant transc ripts. The results with these revertants strongly support our model pr oposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5 ) silenced by hypermethylation. Site-directed mutagenesis studies indi cate that previously proposed ATP-binding and phosphorylation sites ar e not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.