NUCLEOPROTEIN STRUCTURES AND GENOME DUPLICATION

描述（改编自研究者摘要）： 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。这一长期目标 研究的目的是了解使用伽马射线源的启动过程 作为模型系统的抗生素抗性质粒R6 K的复制子。的 R6 K编码的pi蛋白调节复制并自我调节 通过结合含有排列的TGARG序列基序的DNA位点表达 或者在γ ori中插入7个22 bp的同向重复序列（DR），或者插入反向重复序列（DR）。 在其自身基因pir的操纵子中的半重复序列。Pi蛋白具有模块化的 结构; C-末端含有DNA结合结构域，而N-末端 控制pi寡聚化。pi蛋白活性的模型提出， 不同的表面便于DR和IR上的组装。述实验 在具体目标#1中，使用可获得的pi蛋白变体测试该模型， 在复制和转录的控制方面不同于野生型PI。DNA 蛋白质足迹、光交联和基因融合技术将 参与这些调查。在具体目标#2中，研究人员将 鉴定pi结合位点的核苷酸和pi蛋白的氨基酸 通过使用缺失核苷酸技术， 通过基因改变π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 在富含A+ T的γ ori片段中， 大约30个碱基对的上游。具体目标#3将定义 PI与该新位点的结合与通过使用 可用体外复制系统。引发反应也将是 用纯化的组分重构，并将在 这系统.拟议的研究将在分子水平上揭示 一个单一的调节蛋白独立调节 复制和转录。

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.