Roles of Mre11 in Lymphocyte Development and DNA Repair

 描述(申请人提供)：由DNA双链断裂的缺陷反应引起的遗传性疾病以免疫缺陷、骨髓衰竭、淋巴系统恶性肿瘤和发育迟缓为特征。细胞对双链断裂的反应需要专门的DNA损伤识别复合体和核心细胞周期机制之间的快速沟通，但人们对这种重要关系知之甚少。在上一个项目中 在此期间，我们发现了一种意想不到的蛋白质相互作用，这为理解这种关系提供了一个独特的机会。这种相互作用涉及DNA损伤识别机制的核心组件Mre11和细胞周期机制的核心组件细胞周期蛋白依赖性激酶2(CDK2)，前者在共济失调-毛细血管扩张样疾病(ATLD)中发生突变。我们报道了这种相互作用促进了依赖于CDK2的单一底物的磷酸化，首次证明了Mre11在正常的细胞周期中具有作用。在这个提案中，我们提出了Mre11在正常细胞周期中更全局地控制CDK2功能的初步证据。我们的发现表明，当细胞遭受DNA损伤时，这种控制发挥着重要作用。因此，我们将测试最重要的假设，即Mre11与CDK2相互作用并控制CDK2，从而在正常细胞周期和DNA损伤反应之间提供快速切换。我们将确定mre11-CDK2相互作用在不同的生物学背景中所起的作用，例如淋巴细胞发育中的专门化重组，以及更普遍的S时相检查点反应。这里提出的研究利用了我们在前两个项目期间构建的带有Mre11等位基因的小鼠系统，以及我们将开发的新的小鼠模型来直接验证我们的假设。我们在开发独特生物试剂方面的长期投资与我们发现的Mre11-CDK2相互作用相结合，使首席研究员的实验室处于独特的地位，使我们在理解细胞对DNA损伤及其相关疾病的反应方面取得了重大进展。

 DESCRIPTION (provided by applicant): Inherited disorders resulting from defective responses to DNA double strand breaks feature immunodeficiency, bone marrow failure, lymphoid malignancies and developmental delay. Cellular responses to double strand breaks require rapid communication between specialized DNA damage recognition complexes and the core cell cycle machinery, but this important relationship is poorly understood. In the previous project period we identified an unanticipated protein interaction that provides a unique opportunity to make major strides in understanding this relationship. The interaction involves Mre11, a core component of the DNA damage recognition machinery that is mutated in ataxia-telangiectasia like disorders (ATLD), and cyclin dependent kinase 2 (CDK2), a core component of the cell cycle machinery. We reported that the interaction facilitates CDK2 dependent phosphorylation of a single substrate, demonstrating for the first time that Mre11 has roles in the normal cell cycle. In this proposal we present preliminary evidence that Mre11 controls CDK2 functions more globally during the normal cell cycle. Our findings indicate that this control plays an important role when cells undergo DNA damage. Therefore, we will test the overarching hypothesis that Mre11 interacts with and controls CDK2 to provide a rapid switch between the normal cell cycle and the DNA damage response. We will determine roles that Mre11-CDK2 interaction plays is diverse biological contexts such as specialized recombination in lymphocyte development, and S phase checkpoint responses more generally. The studies proposed herein take advantage of murine systems with alleles of Mre11 that we constructed in the previous two project periods, along with new mouse models we will develop to directly test our hypotheses. The combination of our long term investment in the development of unique biological reagents and our discovery of Mre11-CDK2 interaction places the Principle Investigator's laboratory in a unique position to make major strides in our understanding of cellular responses to DNA damage and their associated diseases.