MCL-1 localizes to sites of DNA damage and regulates DNA damage response
MCL-1 localizes to sites of DNA damage and regulates DNA damage response
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DOI：
10.4161/cc.9.14.12354
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发表时间：
2010-07-15
期刊：
影响因子：
4.3
通讯作者：
Duronio, Vincent
中科院分区：
文献类型：
--
作者：
Jamil, Sarwat;Stoica, Cezar;Duronio, Vincent
MCL-1是BCL-2家族的促生存成员，先前已显示在DNA损伤后ATR依赖性Chk 1磷酸化中具有功能。为了进一步阐明这些功能，我们探讨了小鼠胚胎成纤维细胞（MEFs）中缺乏MCL-1引起的DNA损伤反应的可能差异。正如预期的那样，与野生型MEFs相比，Mcl-1（-/-）MEFs在依托泊苷处理后延迟了Chk 1磷酸化。然而，它们对引起G（1）/S检查点反应的羟基脲的反应没有显著差异。此外，在用依托泊苷处理的Mcl-1（-/-）MEFs中，γ-H2 AX的出现延迟。接下来，我们研究了MCL-1是否与其他DNA损伤反应蛋白一起存在于DNA损伤位点。依托泊苷处理的提取物与抗MCL-1抗体的免疫沉淀显示MCL-1与γ-H2 AX以及NBS 1的结合。MCL-1的免疫荧光染色进一步显示DNA损伤后MCL-1和NBS 1的共染色增加。通过使用在基因组中特定位点产生DNA双链断裂的系统，我们证明了MCL-1被直接募集到损伤位点附近。最后，在直接证明MCL-1在允许DNA损伤的适当修复中的重要性时，我们发现两次短暂暴露于依托泊苷的治疗，随后是恢复期，这模拟了依托泊苷使用的临床情况，导致缺乏MCL-1的MEF中染色体异常的更大积累。总之，这些数据表明MCL-1在协调DNA损伤介导的检查点反应中的重要作用，并对MCL-1在维持基因组完整性中的重要性具有广泛的意义。
MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1(-/-) MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G(1)/S checkpoint response, was not significantly different. In addition, appearance of gamma-H2AX was delayed in the Mcl-1(-/-) MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with gamma-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide, followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.