Development of a Novel System to Capture DNA-associated Proteins

 描述（由申请人提供）：在本申请中，我们提出了一种鉴定与特定DNA基因座相关的蛋白质的新方法。我们将这种技术称为DNA-PCap（DNA相关蛋白捕获）。这项新技术能够纯化特定基因组位点附近的蛋白质。这种生物化学方法基于Roux实验室描述的最新技术，该技术允许基于混杂生物素连接酶（BirA*）鉴定与“诱饵”非常接近的蛋白质。在我们作为诱饵的方法中，我们使用RNA引导的DNA结合蛋白（dCas 9）将BirA* 引导至特定的DNA序列。我们在端粒（保护染色体末端的核蛋白复合物）上验证了这种方法。在这个提议中，我们将使用DNA-PCap来鉴定与长散布元件（LINE-1）（活性转座元件）结合并抑制其活化的蛋白质。此外，我们还将应用DNA-PCap技术来鉴定转录调控因子的催化亚基， 端粒酶（hTERT）。这种酶在人体细胞中沉默，但在约90%的人类癌症中重新激活。hTERT再活化的确切机制目前尚不清楚。分离结合hTERT启动子的因子有可能阐明这种关键酶的转录调控机制。该提案的完成将验证DNA-PCap作为一种普遍适用的技术，允许分离与特定DNA元件结合的因子。 公共卫生相关：我们开发了一种新的技术，用于识别与特定DNA序列结合的蛋白质。我们将这种技术称为DNA-PCap（DNA相关蛋白捕获）。我们建议应用DNA-PCap来识别与重复元件和端粒酶启动子相关的因子。

 DESCRIPTION (provided by applicant): In this application, we present a novel method for the identification, of proteins that associate with specific DNA loci. We refer to this technology as DNA-PCap (DNA-associated Protein Capture). The new technology enables the purification of proteins in the vicinity of specific genomic loci. This biochemical approach is based on recent technology described by the Roux laboratory that allows identification of proteins that are in close proximity to a "bait" based on a promiscuous biotin ligase (BirA*). In our approach as bait, we use an RNA- guided DNA binding protein (dCas9) to direct BirA* to specific DNA sequences. We validated this approach at telomeres, nucleo-protein complexes that protect chromosome ends. In this proposal, we will use DNA-PCap to identify proteins that bind to and suppress the activation of the Long Interspersed Elements (LINE-1), active transposable elements. In addition, we will apply DNA-PCap to identify transcriptional regulators of the catalytic subunit of telomerase (hTERT). This enzyme is silenced in human somatic cells, but it is re-activated in ~90% of human cancers. The precise mechanism of hTERT reactivation is currently not known. Isolation of factors that bind to the hTERT promoter has the potential to elucidate the mechanism of transcriptional regulation of this critical enzyme. Completion of this proposal will validate DNA-PCap as a universally applicable technique that allows the isolation of factors that bind to specific DNA elements. PUBLIC HEALTH RELEVANC: We developed a novel technique for the identification of proteins that are bound to specific DNA sequences. We termed this technique DNA-PCap (DNA-associated Protein Capture). We propose to apply DNA-PCap to identify factors that associate with repetitive elements and to the telomerase promoter.