Role of Gene Silencing in the DNA Damage Response

描述（由申请人提供）：在定义控制DNA损伤反应的分子和细胞机制以及该途径如何决定抗癌药物的功效方面已经花费了相当大的努力。这些进展促进了药物的开发，有朝一日可能会帮助医生针对特定疾病定制癌症治疗。DNA损伤反应需要一个协调的核质级联事件，最终聚集在染色质中包装的受损DNA上。已经证明了介导染色质重塑的蛋白质与介导这种损伤反应的蛋白质之间的联系很少。我们研究了DNA损伤诱导的KAP 1磷酸化，KAP 1是所有KRAB-锌指蛋白的专用共阻遏物。该提案将利用三种现有技术，并寻求以一种新的方式应用它们。首先，我们将利用ChIP芯片来确定KAP 1在DNA损伤后是否仍然与已知的结合位点相关联。如果KAP 1在损伤后改变了结合伴侣，那么用全基因组拼接阵列检测到的KAP 1结合的DNA区域应该会改变。如果KAP 1保持与KRAB-ZFP（或另一个尚待确定的锚）相关联，则检测到的与KAP 1结合的位点在损伤后应保持不变。这是第一次使用全基因组平铺阵列来确定关键的染色质重塑因子是否在其专用结合位点附近局部参与DNA损伤反应，或者在受损位点出现时参与全局。该项目的这一部分是Rauscher和Farnham实验室所做工作的逻辑延伸，并利用了双方在KAP 1/KRAB-ZFP范式中取得的重大进展。其次，我们将应用现有的蛋白质半合成方法来创建磷酸化KAP 1的纯池。利用这种修饰的KAP 1作为诱饵，我们将尝试分离一组新的损伤诱导KAP 1相关因子。第三，我们将修改一个串联阵列，目前用于实时成像的转录用于观察和定量的聚集修复因子在双链断裂。我们将首次能够观察单个癌细胞中修复机制的协调组装。此外，它将填补我们知识中的一个重要空白，即染色质在修复之前如何在DNA损伤部位周围快速重塑。该项目代表了Rauscher和Janicki实验室的一个新的令人兴奋的研究方向。基于我们以前的研究和引用的文献，我们认为KAP 1的磷酸化可能在DNA修复机制的某些元件的定位和组装中是至关重要的。该提案不仅试图阐明KAP 1在DNA修复中的作用，而且还确定KAP 1/KRAB-ZFP沉默复合物在DNA损伤后是否发生实质性重组。

DESCRIPTION (provided by applicant): Considerable effort has been expended in defining the molecular and cellular mechanisms governing the DNA damage response and how this pathway determines the efficacy of anti-cancer drugs. These strides have furthered drug development and someday may help physicians tailor their cancer treatment to specific diseases. The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events which ultimately converge on damaged DNA packed in chromatin. Few connections between the proteins that mediate chromatin remodeling and the proteins that mediate this damage response have been demonstrated. We have investigated the DNA damage-induced phosphorylation of KAP1, the dedicated co-repressor for all KRAB-zinc finger proteins. This proposal will utilize three existing technologies and seek to apply them in a novel way. First, we will utilize ChIP-Chip to determine whether the KAP1 remains associated to known binding sites after DNA damage. If KAP1 changed binding partners after damage, then the regions of DNA bound by KAP1 as detected with a whole genome tiling array should become altered. If KAP1 remains associated to KRAB-ZFPs (or another yet to be determined anchor), then the detected sites bound by KAP1 should remain unchanged following damage. This is the first time a whole genome tiling array will be used to determine whether a key chromatin remodeling factor participates in the DNA damage response locally near its dedicated binding site, or globally as damaged sites arise. This part of the project is a logical extension of work done in both Rauscher and Farnham laboratories and takes advantages in major strides made by both parties in the KAP1/KRAB-ZFP paradigm. Second, we will apply an existing method of protein semisynthesis to create a pure pool of phosphorylated KAP1. Using this modified KAP1 as bait, we will attempt to isolate a new group of damage induced KAP1-associating factors. Third, we will modify a tandem array that is currently used for real-time imaging of transcription for use to observe and quantitate the aggregation of repair factors at double stranded breaks. For the first time, we will be capable of observing the coordinated assembly of repair machinery in individual cancer cells. Moreover, it will fill an important gap in our knowledge, namely, how chromatin is rapidly remodeled around sites of DNA damage prior to its repair. This project represents a new and exciting research direction for the Rauscher and Janicki laboratories. Based on our previous studies and the literature cited, we believe that the phosphorylation of KAP1 may be critical in the localization and assembly of some elements of the DNA repair machinery. This proposal not only seeks to clarify the role of KAP1 in DNA repair, but also to determine whether a substantial reorganization of the KAP1/KRAB-ZFP silencing complex occurs after DNA damage.