Origin Recognition Complex at Transcriptional Silencers

描述(申请人提供)：这项研究的目标是全面了解起源识别复合体(ORC)的活动是如何通过与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构体蛋白质复合体，其活性受染色体环境的影响。ORC最为人所知的是它在DNA复制中的关键作用，它标记作为DNA复制起点的染色体位置。除了结合起始点外，ORC还与消音器结合，后者也是依赖ORC发挥功能的小DNA元件。然而，在DNA复制中，消音器的作用效率很低，甚至根本不起作用。相反，沉默分子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体的组装成核来指导抑制(沉默)染色质结构域的形成。这些研究的重点一直是四种酵母沉默因子HMR-E之一，HMR-E是在酵母中一个称为HMRA的座位上建立~4kb沉默染色质结构域所必需且充分的~150bP元件。在上一个资助周期中，我们定义了ORC和特殊辅助蛋白Sir1之间相互作用的分子和结构机制，Sir1定义了ORC在沉默中的作用。我们还了解到，ORC与HMR-E结合的方式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在染色质结构和DNA复制中调节ORC和SIR蛋白活性的机制的新问题。为了解决这些问题，我们将：1.结合遗传学和生化方法来了解ORC-Sir1相互作用是如何调控的；2.使用生化和遗传学方法来定义差异调节ORC功能的ORC-DNA复合体；3.使用全基因组方法来定义在体内调节ORC结合的机制。了解ORC活动对公共健康的影响是巨大的。ORC控制细胞增殖的首要步骤，这一步骤对于控制正常(即组织再生)和不受控制的(即癌症)细胞生长至关重要。此外，以物种特有的方式控制ORC的能力将为开发新型真菌病原体的抑制剂铺平道路。

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.