Molecular and biochemical characterization of xrs mutants defective in Ku80
Molecular and biochemical characterization of xrs mutants defective in Ku80
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DOI：
10.1128/mcb.17.3.1264
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发表时间：
1997-03-01
影响因子：
5.3
通讯作者：
Jeggo, PA
中科院分区：
文献类型：
--
作者：
Singleton, BK;Priestley, A;Jeggo, PA
辐射敏感的CHO突变体属于电离辐射互补组5，其中包括广泛研究的xrs突变体，其基因产物缺陷最近被确定为Ku80， Ku80是Ku蛋白的一个亚基，也是dna依赖性蛋白激酶（DNA-PK）的一个组成部分。一些5组突变体，包括xrs-5和-6，缺乏双链DNA末端结合和DNA- pk活性。在这项研究中，我们在分子和生化水平上研究了额外的xrs突变体。所有突变体的Ku70和Ku80蛋白、末端结合和DNA-PK活性水平均较低或检测不到。只有一个突变体xrs-6可以通过Northern杂交检测到Ku80转录物水平，但大多数其他突变体都可以通过逆转录pcr检测到Ku80 mRNA。两个突变体，xrs-4和-6，由于基因组中影响RNA剪接的Ku80序列发生突变而改变了Ku80转录本，这表明这些突变体的缺陷在于Ku80基因而不是控制其表达的基因。这两个突变体都没有检测到野生型Ku80转录本。由于xrs-4和xrs-6细胞中的突变导致Ku80蛋白严重截断，两者都可能是零突变体的候选者。叠氮胞苷诱导的xrs-4和-6复合体携带野生型和突变型转录本。这些逆转物的结果有力地支持了我们之前提出的模型，即CHO-K1细胞携带一个被超甲基化沉默的Ku80基因（XRCC5）拷贝。位点导向诱变研究表明，先前提出的atp结合位点和磷酸化位点并不需要Ku80活性，而n端超过前7个氨基酸的缺失会导致严重的活性丧失。
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 he 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.