Role of Gene Silencing in the DNA Damage Response

基因沉默在 DNA 损伤反应中的作用

• 批准号: 8241122
• 负责人: FRANK JOSEPH RAUSCHER III
• 金额: $ 26.94万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8241122
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Amino Acids; Antineoplastic Agents; Architecture; Area; BRCA1 gene; Binding; Binding Sites; Boxing; ChIP-on-chip; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Complex; DNA; DNA Binding; DNA Damage; DNA Repair; DNA strand break; Data; Disease; Double Strand Break Repair; Elements; Enzymes; Event; Family; Gene Silencing; Genetic Transcription; Genome; Human; Image; Individual; Ionizing radiation; Knowledge; Laboratories; Lesion; Link; Literature; Malignant Neoplasms; Maps; Mediating; Methods; Molecular; Mus; Nonhomologous DNA End Joining; Pathway interactions; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physicians; Play; Proteins; Repair Complex; Research; Role; Serine; Site; Technology; Testing; Therapeutic; Time; Work; Zinc Fingers; base; cancer cell; cancer therapy; cell injury; chromatin remodeling; drug development; follow-up; gene repression; homologous recombination; human ZNF45 protein; insight; irradiation; knowledge base; member; novel; recombinational repair; repaired; response; scaffold; ubiquitin-protein ligase

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.

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大量的努力。这些进步进一步推动了药物开发，有朝一日可能会帮助医生针对特定疾病调整癌症治疗方法。 DNA 损伤反应需要协调的核-细胞质级联事件，最终汇聚到染色质中受损的 DNA 上。介导染色质重塑的蛋白质和介导这种损伤反应的蛋白质之间几乎没有联系。我们研究了 DNA 损伤诱导的 KAP1 磷酸化，KAP1 是所有 KRAB 锌指蛋白的专用共抑制因子。该提案将利用三项现有技术并寻求以新颖的方式应用它们。首先，我们将利用 ChIP-Chip 来确定 DNA 损伤后 KAP1 是否仍然与已知的结合位点相关。如果 KAP1 在损伤后改变了结合伴侣，那么用全基因组平铺阵列检测到的 KAP1 结合的 DNA 区域应该会发生改变。如果 KAP1 仍然与 KRAB-ZFP（或另一个尚未确定的锚）相关，则检测到的由 KAP1 结合的位点在损坏后应保持不变。这是第一次使用全基因组平铺阵列来确定关键染色质重塑因子是否参与其专用结合位点附近的局部 DNA 损伤反应，或者当受损位点出现时全局参与 DNA 损伤反应。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 KAP1/KRAB-ZFP 范例中取得的重大进展。其次，我们将应用现有的蛋白质半合成方法来创建磷酸化 KAP1 的纯库。使用这种修饰的 KAP1 作为诱饵，我们将尝试分离一组新的损伤诱导 KAP1 相关因子。第三，我们将修改目前用于转录实时成像的串联阵列，用于观察和定量双链断裂处修复因子的聚集。我们将第一次能够观察到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 损伤位点周围快速重塑。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。根据我们之前的研究和引用的文献，我们认为 KAP1 的磷酸化可能对于 DNA 修复机制某些元件的定位和组装至关重要。该提案不仅旨在阐明KAP1在DNA修复中的作用，而且还确定DNA损伤后KAP1/KRAB-ZFP沉默复合物是否发生实质性重组。