Mechanisms of chromosome-scale signal propagation

 描述（由申请人提供）：该项目的总体目标是确定细胞如何在大的染色体距离上传递染色体断裂信号。DNA双链断裂（DSB）是对基因组完整性的危险损害，因为它们可能导致染色体重排和染色体不稳定性，这两者与癌症进展以及出生缺陷密切相关。在同时发生多个DSB的情况下，基因组不稳定性的风险急剧放大，如放射治疗和许多形式的化疗。然而，至少在某些情况下，细胞能够有效地协调多个并发DSB的修复。最突出的例子是减数分裂，当生殖细胞在其大多数基因组中引入数百个编程的DSB时。减数分裂DSB修复的一个关键特征是它在染色体水平上协调，使得一个DSB处的修复决定以染色体自主的方式传递到在同一染色体上发生在很远距离处的DSB。这种交流发生的机制基本上是未知的，但将为细胞如何科普大量染色体损伤提供重要的新见解。初步分析减数分裂DNA损伤信号在有性繁殖酵母酿酒酵母揭示了几个信号，似乎明显传播沿着减数分裂染色体减数分裂DSB形成。我们假设，这些信号形成的通信设备，允许减数分裂细胞沟通DSB修复决策的一部分。这些信号有几种不同的形式，包括蛋白磷酸化的传播和染色体结构的变化，并表现出时间和空间的差异，表明它们可能传达减数分裂DSB修复过程的不同方面。为了确定这些信号的减数分裂作用，染色体信号传导和DSB修复的动力学将通过遗传学和超分辨率显微镜进行分析，利用一种新的条件性核耗竭方法，该方法允许阶段特异性敲除通常的多效性修复因子。此外，将使用细胞学、生物化学和修复中间体的物理分析来分析信号整合。最后，该提案将缩小一个主要的技术差距，开发一种方法来绘制整个基因组的DSB修复中间体。总之，这些分析将首次深入了解染色体信号传播控制DNA修复的机制，并为理解导致出生缺陷和癌症的DSB修复错误开辟新的途径。

 DESCRIPTION (provided by applicant): The overall goal of this project is to determine how cells communicate chromosome break signals across large chromosomal distances. DNA double-strand breaks (DSBs) are dangerous insults to genome integrity because of their potential to cause chromosome rearrangements and chromosome instability, both of which are strongly associated with cancer progression as well as birth defects. The risk of genome instability is dramatically amplified in situations where multiple DSBs occur at the same time, as is the case with radiotherapy and many forms of chemotherapy. However, at least under certain circumstances, cells are able to efficiently orchestrate the repair of multiple concurrent DSBs. The most prominent example is meiosis, when germ cells introduce hundreds of programmed DSBs across most of their genomes. A key feature of meiotic DSB repair is that it is coordinated at a chromosomal level, such that repair decisions at one DSB are transmitted in a chromosome- autonomous way to DSBs that occurred a large distance away on the same chromosome. The mechanism by which such communication occurs is essentially unknown, but would provide important new insights into how cells cope with massive chromosomal insults. Preliminary analysis of meiotic DNA damage signaling in the sexually reproducing yeast Saccharomyces cerevisiae revealed several signals that appeared to visibly propagate along meiotic chromosomes following meiotic DSB formation. We hypothesize that these signals form part of the communication apparatus that allows meiotic cells to communicate DSB repair decisions. The signals take several different forms, including propagation of protein phosphorylation and changes in chromosome structure, and exhibit temporal and spatial differences, suggesting that they may communicate different aspects of the meiotic DSB repair process. To determine the meiotic roles of these signals, the dynamics of chromosomal signaling and DSB repair will be analyzed by genetics and super resolution microscopy, taking advantage of a novel conditional nuclear depletion approach that allows stage-specific knock-downs of the often pleiotropic repair factors. In addition, signal integration will be analyzed usig cytology, biochemistry, and physical analysis of repair intermediates. Finally, the proposal will close a major technological gap with the development of a method to map DSB repair intermediates across the entire genome. Together, these analyses will provide first insights into the mechanisms of chromosomal signal propagation controlling DNA repair, and open new avenues for understanding the errors in DSB repair that cause birth defects and cancer.