Origin Recognition Complex at Transcriptional Silencers

描述（由申请人提供）：本研究的目的是全面了解起源识别复合体（ORC）的活性是如何通过与DNA和辅助蛋白的相互作用来调节的。ORC是一种进化上保守的异质蛋白复合物，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，它标记作为DNA复制起点的染色体位点。除了结合起源外，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，沉默基因通过使结合和修饰核小体的SIR（沉默信息调节）蛋白的特殊蛋白质复合物组装成核，直接形成抑制性（沉默）染色质结构域。这些研究的重点是酵母四种沉默基因之一，HMR-E，一个约150 bp的元件，足以在酵母中一个名为HMRa的位点上建立一个约4 kb的沉默染色质结构域。在上一个授权周期中，我们定义了控制ORC与特殊辅助蛋白Sir1之间相互作用的分子和结构机制，Sir1定义了ORC在沉默子中的作用。我们还了解到，ORC与HMR-E的结合与几个复制起点不同，这些差异有助于HMR-E在形成沉默染色质方面的积极作用，以及它实际上无法作为DNA复制起点发挥作用。这些和其他数据提出了关于ORC和SIR蛋白在染色质结构和DNA复制中调节活性的机制的新问题。为了解决这些问题，我们将：1。结合遗传和生化方法来辨别ORC-Sir1相互作用是如何被调节的；2. 使用生化和遗传方法定义ORC- dna复合物，差异调节ORC功能；3. 使用全基因组方法定义体内调节ORC结合的机制。了解ORC活动对公共卫生的影响是巨大的。ORC控制着细胞增殖的第一步，这是控制正常（即组织再生）和不受控制（即癌症）细胞生长所必需的一步。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.