TELOMERE DYSFUNCTION: A TOOL FOR THE STUDY OF NOVEL DNA DAMAGE RESPONSE FACTORS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 DNA损伤反应（DDR）途径在确保基因组信息的完整性方面起着重要的肿瘤抑制作用。事实上，影响这一途径的组分的突变在人类癌症中很常见。尽管在DNA损伤领域进行了广泛的研究，但这一过程中的几个步骤仍然知之甚少，这表明这一途径中的关键参与者尚未被确定。特别是，缺乏表征的是细胞检测DNA损伤的机制，这是DNA损伤反应启动的初始和关键步骤。两个主要的限制阻碍了该领域的研究：i）由常规DNA损伤诱导剂诱导的DNA损伤位点的不可预测的位置，ii）缺乏用于分离与给定DNA底物相关的蛋白质的技术。我将克服这些限制，利用：i）端粒功能障碍作为一种工具，诱导DNA损伤的特定基因组位点，ii）一种新的技术开发的分离蛋白质与特定的染色质位点称为蛋白质组学分离染色质片段（PICh）。结合这两种方法，我将能够在定义的基因组位点上诱导DNA损伤，以分离和鉴定定位于DNA损伤位点的DNA损伤机制的组分。这种方法是高度创新的，并有可能确定哺乳动物细胞中的DNA损伤途径的新组件。鉴于在癌症中发现影响该途径组分的突变的频率，我们预计我们的研究结果将在基因组稳定性和癌症生物学领域具有重要意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The DNA damage response (DDR) pathway plays an essential tumor suppressive role ensuring the integrity of genomic information. Indeed, mutations affecting components of this pathway are common in human cancer. Despite extensive research in the DNA damage field several steps in this process remain poorly understood suggesting that crucial players in this pathway have yet to be identified. In particular, poorly characterized is the mechanism by which cells detect DNA lesions, which is the initial and crucial step for DNA damage response initiation. Two major limitations have hindered the research in this field: i) the unpredictable location of DNA damage sites induced by conventional DNA damage-inducing agents, ii) the lack of a technique for the isolation of proteins associated with a given DNA substrate. I will overcome these limitations by taking advantage of: i) telomere dysfunction as a tool to induce DNA damage to specific genomic loci, ii) a novel technique developed to isolate protein associated with specific chromatin loci termed Proteomics of Isolated Chromatin segments (PICh). Combining these two approaches I will be able to induce DNA damage on defined genomic loci in order to isolate and identify the components of the DNA damage machinery that localize to DNA damage sites. This approach is highly innovative and has the potential of identifying novel components of the DNA damage pathway in mammalian cells. Given the frequency by which mutations affecting components of this pathway are found in cancer we anticipate that the results of our study will be of great relevance in the field of genomic stability and cancer biology.