Origin Recognition Complex at Transcriptional Silencers

描述（由申请人提供）：本研究的目标是全面了解起源识别复合物（ORC）的活性如何通过与DNA和辅助蛋白的相互作用进行调节。ORC是一种进化上保守的异聚体蛋白复合物，其活性受染色体环境的影响。ORC以其在DNA复制中的关键作用而闻名，其中它标记作为DNA复制起点的染色体位点。除了结合起点，ORC还结合沉默子，沉默子也是依赖ORC发挥功能的小DNA元件。然而，沉默子在DNA复制中效率低下或根本不起作用。相反，沉默子通过使结合和修饰核小体的SIR（沉默信息调节子）蛋白的专门蛋白质复合物的组装成核来指导抑制（沉默）染色质结构域的形成。这些研究的焦点是四种酵母沉默子之一，HMR-E，一个在酵母中称为HMRa的基因座处建立约4kb沉默染色质结构域所必需且足够的约150 bp元件。在上一个资助周期中，我们定义了ORC和专门的辅助蛋白Sir 1之间相互作用的分子和结构机制，Sir 1定义了ORC在沉默剂中的作用。我们还了解到ORC与HMR-E的结合不同于几个复制起点，这些差异有助于HMR-E在形成沉默染色质中的积极作用及其实际上不能作为DNA复制起点。这些和其他数据提出了新的问题，有关的机制，调控的ORC和SIR蛋白的活性，在染色质结构和DNA复制。为了解决这些问题，我们将：1。结合联合收割机的遗传和生化方法来辨别如何ORC-Sir 1相互作用的调节; 2.使用生物化学和遗传学方法来定义差异调节ORC功能的ORC-DNA复合物; 3.使用全基因组方法来定义调节ORC体内结合的机制。了解ORC活动的公共卫生意义是巨大的。ORC控制细胞增殖中的首要步骤，这是操纵正常（即，组织再生）和不受控制（即，癌）细胞生长。此外，以物种特异性方式控制ORC的能力将为开发新兴真菌病原体类别的抑制剂铺平道路。

DESCRIPTION (provided by applicant): The goal of this research is to gain a comprehensive understanding of how the activities of the origin recognition complex (ORC) are regulated by interactions with DNA and accessory proteins. ORC is an evolutionarily conserved heteromeric protein complex whose activities are affected by chromosomal context. ORC is best known for its pivotal role in DNA replication where it marks chromosomal sites that serve as DNA replication origins. In addition to binding origins, ORC also binds to silencers that are also small DNA elements that depend on ORC for their function. However, silencers function inefficiently or not at all in DNA replication. Instead, silencers direct formation of repressive (silent) chromatin domains by nucleating the assembly of a specialized protein complex of SIR (silent information regulator) proteins that bind and modify nucleosomes. The focus of these studies has been one of four yeast silencers, HMR-E, a ~150 bp element necessary and sufficient to establish a ~4 kb silent chromatin domain at a locus in yeast called HMRa. In the last grant cycle, we defined the molecular and structural mechanisms governing an interaction between ORC and the specialized accessory protein Sir1 that defines ORC's role at silencers. We also learned that ORC binds HMR-E differently from several replication origins, and that these differences contribute to both HMR-E's positive role in forming silent chromatin and its virtual inability to function as a DNA replication origin. These and other data have raised new questions concerning the mechanisms that modulate the activities of ORC and SIR proteins in both chromatin structure and DNA replication. To address these questions we will: 1. Combine genetic and biochemical approaches to discern how the ORC-Sir1 interaction is regulated; 2. Use biochemical and genetic approaches to define ORC-DNA complexes that differentially modulate ORC function; 3. Use whole-genome approaches to define mechanisms that modulate ORC binding in vivo. The public health implications of understanding ORC activity are immense. ORC controls the premiere step in cell proliferation, a step essential for manipulating both normal (i.e., tissue regeneration) and uncontrolled (i.e., cancer) cell growth. Further, the ability to control ORC in species-specific manners will pave the way for developing inhibitors of the emerging class of fungal pathogens.