Interrogation of systems level mechanisms controlling DNA repair processes

描述(由申请人提供)：一个完整的生物系统的综合模型将能够在面对新的遗传和环境扰动时预测表型结果。对细胞系统的这种预测能力，如DMA修复，将对遗传疾病的早期发现和诊断产生巨大影响，并最终在设计预防和/或治疗方法方面产生巨大影响。然而，由于其巨大的复杂性，充分理解真核细胞的DMA修复系统是一个艰巨的挑战，特别是考虑到系统方法还不够成熟，不足以对这种复杂的过程进行数学建模。在分子水平上，真核生物的遗传信息处理以一种简化的方式被它们的进化祖先古生菌所反映。在盐生杆菌NRC-1中，我们有一个简单但强大的-2400个基因的模型系统，其中可以描述系统方法的潜在普遍原理。这种嗜盐古细菌通常可以减轻高盐度、紫外线辐射和干燥-再水化循环造成的损害。我们将使用这个模型系统来处理这样的假设，即在暴露于DMA损伤事件(如紫外线照射)后，控制细胞命运的决策过程是由强大的基因调控网络介导的，这些网络同时处理关于撞击辐射的光谱特征、DMA损伤的性质和程度以及细胞携带对日周期的潜在准备作用的信息。这一假说是基于两个主要的观察结果：a.在紫外线反应的系统分析中，尽管具有非凡的抗性，但并非该微生物中所有预期的修复基因都对辐射或损伤做出反应；b.许多这些损伤反应迟钝的修复基因确实对昼夜夹带做出反应。在基本水平上，这表明细胞在照射时的驻留状态可能会影响所引起的反应的类型。系统化的方法非常适合于去除这一决策过程背后的网络。具体地说，我们将测量野生型和突变菌株(传感器、信号转导、调节器和修复蛋白缺陷)在入射辐射、造成的损伤类型和辐射时细胞的驻留状态组合变化下的动态全球变化(损伤程度、mRNA/蛋白质水平、蛋白质-蛋白质和蛋白质-DNA相互作用)。通过对这些不同系统水平数据的综合分析，我们将从统计学上了解扰动诱导的基因调控网络的重新布线，该网络将环境扰动(输入)处理为表型(输出)，即用于修复的调控机制的预测模型。这个基本模型将作为设计系统方法的模板，以模拟真核细胞修复过程的更高复杂性。

DESCRIPTION (provided by applicant): A comprehensive model of a complete biological system will enable prediction of phenotypic outcomes in face of novel genetic and environmental perturbations. Such predictive power for cellular systems, such as DMA repair, will have tremendous impacts on early detection and diagnosis of genetic disorders and, ultimately in designing preventive and/or curative treatments. However, the full understanding of eukaryotic DMA repair systems presents a daunting challenge due to their enormous complexity, especially given that systems approaches are not yet sufficiently mature to mathematically model such complex processes. At a molecular level, eukaryotic genetic information processing is mirrored in a simplified manner by their evolutionary ancestors, the archaea. In Halobacterium NRC-1 we have a simple yet powerful model system of -2400 genes in which the underlying generalized principles of a systems approach can be delineated. This halophilic archaeon routinely mitigates damage from high salinity, UV radiation and desiccation-rehydration cycles. We will use this model system to address the hypothesis that the decision making process governing cell fate after exposure to DMA damaging events such as UV irradiation is mediated by robust gene regulatory networks that simultaneously process information on spectral characteristics of the impinging radiation, the nature and extent of DMA damage and the potential preparative role of cellular entrainment to diurnal cycles. This hypothesis is motivated by two main observations: a. in systems analysis of UV response, although extraordinarily resistant, not all expected repair genes in this microbe responded to irradiation or damage; b. many of these damage-unresponsive repair genes did, however, respond to day-night entrainment. At a fundamental level this suggested that the resident state of the cell at time of irradiation may influence the type of response elicited. A systems approach is ideally suited to delinate the networks underlying this decision making process. Specifically, we will measure dynamic global changes (degree of damage, mRNA /protein levels, protein-protein and protein-DNA interactions) in wild type and mutant strains (defective in sensors, signal transducers, regulators and repair proteins) subjected to combinatorial changes in incident radiation, type of damage inflicted and resident state of the cell at the time of radiation. Through an integrated analysis of these diverse systems level data we will statistically learn perturbation-induced rewiring of a gene regulatory network that processes environmental perturbations (input) into phenotype (output), i.e. a predictive model for regulatory mechanisms for repair. This basic model will serve as a template for designing systems approaches to model higher complexities of eukaryotic repair processes.