WASH复合体与Ku蛋白的相互作用及其在DNA双链断裂修复中的作用与机制

The WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) is the important Actin nucleation promoting factors in the cytoplasm. The WASH complex is distributed in both cytoplasm and nucleus, but the function of the WASH complex in the nucleus is not clear. By mass spectrometry, co-immunoprecipitation and immunofluorescence, we found that WASH/FAM21, the core members of WASH complex, had interaction with Ku70/Ku80 in the nucleus. It is known that the Ku70/Ku80 heterodimer is the key molecule to initiate the NHEJ (Non-homologous end joining) pathway. We found that knockdown of WASH inhibited the formation of 53BP1 foci which is the DSB (double strand break) marker. This suggests that the nuclear WASH complex may play a role in DNA DSB repair. Therefore this study aims to elucidate the molecular basis of the interaction between WASH complex and Ku70/Ku80, and explore the role and mechanism of WASH in DNA DSB repair. This study would deepen the understanding of the nuclear WASH complex function and DNA damage repair mechanism.

WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog)是细胞浆中重要的促肌动蛋白成核因子。WASH复合体在细胞浆和细胞核内均有分布，但目前WASH复合体在细胞核内的功能尚不明确。利用质谱鉴定、免疫共沉淀和免疫荧光技术，我们发现在细胞核内WASH复合体核心成员WASH/FAM21与Ku70/Ku80存在相互作用。已知Ku70/Ku80二聚体是启动DNA非同源末端连接途径的关键分子。我们发现沉默WASH会抑制DNA双链断裂标志分子53BP1的foci形成。这提示在细胞核内WASH复合体可能在DNA双链断裂修复中发挥作用。因此本项目拟分析WASH复合体与Ku70/Ku80相互作用的分子基础，探索WASH在DNA双链断裂修复方面的作用和机制，以加深对细胞核内WASH复合体功能和DNA损伤修复机制的理解。

作为一种促成核因子，WASH通过激活Arp2/3调控内体的分选和转运。越来越多的证据支持WASH存在于细胞核中，并且在细胞核内WASH调节基因转录和自噬。因此细胞核内WASH的功能和机制是WASH研究领域的关键科学问题之一。在本研究中，我们确证了WASH/FAM21与多种非同源末端连接（NHEJ）因子Ku70、Ku80和DNA-PKcs的相互作用，并且发现Ku70/Ku80能够募集WASH/FAM21至DNA双链断裂（DSB）位点。重要的是，WASH缺失增加了细胞对etoposide诱导的DSBs的敏感性，损害了细胞的修复能力，降低了NHEJ的效率。其机制在于WASH的缺失迟滞了DNA损伤后H2AX和KAP1的磷酸化，抑制了染色体的解凝和NHEJ因子XLF和DNA-ligase 4向DSBs的募集。此外，WASH在DNA修复中的功能依赖于其保守的VCA结构域和Arp2/3激活。总之，我们的工作揭示了WASH通过NHEJ通路调控DNA修复的作用及其分子机制。本课题的完成为解析细胞核内WASH的功能和进一步理解DNA修复的机制提供了新的视角和切入点。