Origin Recognition Complex at Transcriptional Silencers

转录沉默子的起源识别复合体

• 批准号: 7904366
• 负责人: Catherine A Fox
• 金额: $ 20.93万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904366
• 关键词: Address; Affect; Affinity; Architecture; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biological; Biological Assay; Cancer Cell Growth; Cell Proliferation; Cells; Chemicals; Chromatin; Chromatin Structure; Complex; Computer Simulation; DNA; DNA Binding; DNA Sequence; DNA biosynthesis; DNA replication origin; Data; Data Set; Elements; Foundations; Genetic; Genome; Goals; Grant; Growth; Histones; In Vitro; Learning; Maps; Mating Types; Mediating; Methylation; Molecular; Nucleosomes; Partner in relationship; Peptides; Protein Binding; Proteins; Public Health; Recruitment Activity; Replication Initiation; Replication Origin; Research; Resolution; Role; Saccharomycetales; Sequence Analysis; Site; Tail; Temperature; Testing; Yeasts; base; crosslink; density; design; in vivo; inhibitor/antagonist; mutant; origin recognition complex; pathogen; protein complex; research study; tissue regeneration; tool; virtual

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.

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蛋白复合物，其活性受染色体环境影响。 ORC 因其在 DNA 复制中的关键作用而闻名，它标记了作为 DNA 复制起点的染色体位点。除了结合起点外，ORC 还与沉默子结合，沉默子也是依赖于 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（沉默信息调节器）蛋白的特殊蛋白复合物的组装成核来直接形成抑制性（沉默）染色质结构域。这些研究的重点是四种酵母沉默子之一，即 HMR-E，这是一种约 150 bp 的元件，它是在酵母中称为 HMRa 的基因座上建立约 4 kb 沉默染色质结构域所必需和充分的。在上一个资助周期中，我们定义了控制 ORC 和特殊辅助蛋白 Sir1 之间相互作用的分子和结构机制，该辅助蛋白定义了 ORC 在消音器中的作用。我们还了解到，ORC 与几个复制起点的 HMR-E 结合方式不同，这些差异既导致 HMR-E 在形成沉默染色质方面发挥积极作用，又导致其实际上无法充当 DNA 复制起点。这些数据和其他数据提出了有关调节 ORC 和 SIR 蛋白在染色质结构和 DNA 复制中的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了解 ORC-Sir1 相互作用是如何受到调节的； 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 活动对公共卫生的影响是巨大的。 ORC 控制细胞增殖的首要步骤，这是操纵正常（即组织再生）和不受控制（即癌症）细胞生长所必需的步骤。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。