NUCLEOPROTEIN STRUCTURES AND GENOME DUPLICATION

核蛋白结构和基因组复制

• 批准号: 6385729
• 负责人: MARCIN S FILUTOWICZ
• 金额: $ 27.19万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385729
• 关键词: DNA binding protein; DNA footprinting; DNA replication; chimeric proteins; crosslink; gel mobility shift assay; gene expression; genetic promoter element; genetic transcription; methylation; nucleoproteins; protein sequence; protein structure function; reporter genes

DESCRIPTION (adapted from investigator's abstract): Recognition of the replication origin by initiator protein is the central event regulating DNA replication in diverse biological systems. The long term objective of this research is to understand the initiation process using the gamma origin replicon of the antibiotic resistance plasmid R6K as a model system. The R6K-encoded pi protein regulates replication and autoregulates its own expression by binding to DNA sites containing a TGARG sequence motif arranged either into seven 22-bp direct repeats (DRs) in the gamma ori, or into inverted half-repeats in the operator of its own gene pir. Pi protein has a modular structure; the C-terminus contains the DNA binding domain while the N-terminus controls pi oligomerization. The model of pi protein activity proposes that its distinct surfaces facilitate assemblies on DRs and IRs. Experiments described in Specific Aim #1 test this model using available variants of pi protein that differ from wild type pi in the control of replication and transcription. DNA and protein footprinting, photo-crosslinking and genetic fusion techniques will be employed in these investigations. In Specific Aim #2 the investigators will identify the nucleotides in pi binding sites and the amino acids in pi protein that intimately contact one another by using missing nucleotide technology and by genetically altering the DNA binding properties of pi. The investigators have recently identified a novel, non-TGAGRG pi binding site in the A+T-rich segment of gamma ori and have mapped the start of DNA synthesis approximately 30-bp upstream from this site. Specific Aim #3 will define the relationship between pi binding to this novel site and initiation by using an available in vitro replication system. The priming reaction will also be reconstituted with purified components and the effect of pi will be examined in this system. The proposed studies will reveal at a molecular level the mechanisms by which a single regulatory protein independently regulates replication and transcription.

描述（改编自研究者的摘要）：对 起始蛋白的复制起点是调节 DNA 的中心事件 在不同的生物系统中复制。本次活动的长远目标 研究是利用伽玛原点来了解引发过程 抗生素抗性质粒 R6K 的复制子作为模型系统。这 R6K 编码的 pi 蛋白调节复制并自动调节自身 通过与含有排列的 TGARG 序列基序的 DNA 位点结合来表达 或者进入 gamma ori 中的 7 个 22-bp 直接重复序列 (DR)，或者进入反向序列 在其自身基因 pir 的操作符中半重复。 Pi 蛋白具有模块化 结构; C 末端包含 DNA 结合域，而 N 末端包含 控制 pi 寡聚化。 pi 蛋白活性模型提出， 不同的表面有利于 DR 和 IR 上的组装。实验描述 在特定目标 #1 中，使用 pi 蛋白的可用变体测试该模型 与野生型pi的不同之处在于复制和转录的控制。脱氧核糖核酸 蛋白质足迹、光交联和基因融合技术将 受雇参与这些调查。在具体目标#2 中，调查人员将 识别 pi 结合位点中的核苷酸和 pi 蛋白中的氨基酸 通过使用缺失核苷酸技术彼此密切接触， 通过基因改变 pi 的 DNA 结合特性。 研究人员最近发现了一个新的非 TGAGRG pi 结合位点 位于 gamma ori 富含 A+T 的片段中，并绘制了 DNA 合成起始点的图谱 该位点上游约 30-bp。具体目标 #3 将定义 pi 与该新位点的结合与通过使用 可用的体外复制系统。引发反应也将是 用纯化的成分重构，并且将在以下中检查 PI 的效果 这个系统。拟议的研究将在分子水平上揭示 单个调节蛋白独立调节的机制 复制和转录。