Role of EMSY protein complexes in the FA DNA repair pathway

 描述(申请人提供)：像Fanconi贫血(FA)这样的基因组不稳定疾病是人类对癌症易感性的罕见遗传模式。FA是一种多基因疾病，在细胞水平上以染色体异常为特征，并对复制阻断DNA损伤，特别是DNA链间交联(DNA ICL)高度敏感。FA途径的一个中心成员是FANCD2蛋白，它被直接招募到DNA ICL和停滞的复制叉子中。越来越多的证据表明，FANCD2然后招募其他FA和非FA DNA修复因子，这些因子使用同源重组(HR)修复机制来介导复制阻断损伤的移除，如DNA ICL，并促进复制分叉恢复。基于我们的新结果，我们预测一个名为EMSY的蛋白质将成为FANCD2的一个新的功能伙伴。EMSY是否在任何细胞DNA损伤反应中发挥作用尚不清楚。我们发现EMSY以DNA损伤的方式招募到染色质中，并且严格依赖于FANCD2。值得注意的是，EMSY缺陷细胞与FANCD2缺陷细胞一样对DNA ICL和复制压力高度敏感。根据我们的初步结果，我们假设EMSY和FANCD2形成了一种新的蛋白质复合体，促进HR依赖的DNA ICL修复和HR介导的复制分叉恢复。我们将验证这一假说如下：目的1：确定EMSY/FANCD2在HR介导的DNA ICL修复中的作用。我们将测试EMSY和FANCD2是否协同作用，促进细胞DNA ICL抗性，并在DNA ICL相关DNA双链断裂(DSB)的HR修复过程中支持分子步骤。目的2：确定EMSY/FANCD2在HR介导的复制分叉恢复中的作用。我们将测试EMSY和FANCD2是否协同作用，以促进细胞对Ahidiclin触发的复制分叉停滞的抵抗，并在HR因子介导的复制分叉保护和重新启动过程中支持分子步骤。此外，我们将测试六个已知的FANCD2患者突变是否通过中断FANCD2/EMSY复合体的形成而导致复制分叉恢复中的细胞缺陷。这些方法将阐明目前尚未确定的EMSY蛋白如何在细胞DNA损伤反应中发挥作用，并揭示FA途径相关DNA修复网络中新的复杂性。我们希望我们的发现将大大有助于理解FA途径在维持基因组稳定和保护细胞免受突变事件中的作用。这反过来将影响对癌症易感性和化疗治疗策略的研究。

 DESCRIPTION (provided by applicant): Genomic instability diseases like Fanconi Anemia (FA) represent rare genetic models for human susceptibility to cancer. FA is a multi-gene disease characterized on the cellular level by chromosomal abnormalities and a hypersensitivity to replication-blocking DNA lesions, particularly DNA interstrand crosslinks (DNA ICLs). A central member of the FA pathway is the FANCD2 protein that is directly recruited to DNA ICLs and stalled replication forks. Accumulating evidence suggests that FANCD2 then recruits other FA and non-FA DNA repair factors that use homologous recombination (HR) repair mechanisms to mediate the removal of replication- blocking lesions such as DNA ICLs, and to promote replication fork recovery. Based on our new results, we predict a protein named EMSY to be a novel functional partner of FANCD2. Whether EMSY functions in any cellular DNA damage responses is not known. We discovered that EMSY is recruited to chromatin in a DNA damage- and strictly FANCD2-dependent manner. Strikingly, EMSY-deficient cells are as hypersensitive to DNA ICLs and to replication stress as FANCD2-deficient cells. Based on our preliminary results, we hypothesize that EMSY and FANCD2 form a novel protein complex that promotes HR- dependent DNA ICL repair and HR-mediated replication fork recovery. We will test this hypothesis as follows: Aim 1: Determine the role of EMSY/FANCD2 in HR-mediated DNA ICL repair. We will test if EMSY and FANCD2 act in concert to promote cellular DNA ICL resistance and to support molecular steps during the HR- repair of DNA ICL-associated DNA double strand breaks (DSBs). Aim 2: Determine the role of EMSY/FANCD2 in HR-mediated replication fork recovery. We will test if EMSY and FANCD2 act in concert to promote cellular resistance to aphidicolin-triggered replication fork stalling and to support molecular steps during HR factor-mediated protection and restart of replication forks. Additionally, we will test if six known FANCD2 patient mutations cause cellular deficiencies in replication fork recovery by interrupting FANCD2/EMSY complex formation. These approaches will elucidate how the currently uncharacterized EMSY protein function in the cellular DNA damage response and reveal a new layer of complexity within the FA pathway-associated DNA repair network. We expect our findings to significantly contribute to an understanding of the FA pathway's role in maintaining genomic stability and in protecting cells from mutagenic events. This in turn will impact research on cancer predisposition and chemotherapeutic treatment strategies.