Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair
Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair
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DOI：
10.1074/jbc.m106295200
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发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
Takeda, S
中科院分区：
文献类型：
--
作者：
Fukushima, T;Takata, M;Takeda, S
脊椎动物中存在两条主要的互补双链断裂(DSB)修复途径，即涉及RAD54的同源重组(HR)和需要DNA依赖的蛋白激酶(DNA-PK)的非同源末端连接。DNA-PK包括一个催化亚基(DNA-PKcs)和一个与DNA结合的Ku70和Ku80异源二聚体。为了确定单个DNA-PK组分在DSB修复中的活性，我们在鸡DT40细胞中靶向了DNA-PKcs基因。DNA-PKcs缺失导致DSB修复缺陷，该缺陷意外地被KU70中断抑制。我们以前已经证明，当姐妹染色单体可用于HR修复时，KU70的遗传消融在S-G(2)期细胞上赋予RAD54依赖的辐射抗性。为了验证Ku70对HR的直接干扰是否可以解释Ku70(-/-)/DNA-PKcs(-/-/-)辐射抗性，我们直接监测了Ku-和DNA-PKcs缺陷细胞的HR活性。在没有Ku的情况下，I-SCEI限制性内切酶诱导的染色体内HR频率增加，但DNA-PKcs的频率不变。值得注意的是，靶向RAD54的Ku 70(-/-)或DNA-PKcs(-/-/-)细胞中的HR活性的丧失导致了极端的辐射敏感性，这表明Ku70的相对辐射抗性是由于HR依赖的修复途径所致。我们的研究结果表明，Ku可以干扰HR介导的DSB修复，可能与HR竞争DSB识别。
Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku 70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.