Developmental Control of DNA Replication in Caulobacter

柄杆菌 DNA 复制的发育控制

• 批准号: 7982524
• 负责人: LUCILLE SHAPIRO
• 金额: $ 30.14万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7982524
• 关键词: Actins; Antibiotics; Antisense RNA; Behavior; Binding; Biochemical; Boron; Caulobacter; Cell Cycle; Cell Cycle Regulation; Cells; Centromere; Chromosome Segregation; Chromosomes; Complex; DNA; DNA Methylation; DNA Replication Timing; DNA Sequence; DNA biosynthesis; Development; Event; Family; Free Ribosome; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Transcription; Goals; Image; In Vitro; Individual; Life; Link; Mediating; Methylation; Movement; Phase; Positioning Attribute; Proteins; Replication Initiation; Resolution; Rest; Role; Running; Structure; System; Time; Transcript; Transcriptional Regulation; base; cdc Genes; chromosome replication; design; in vivo; inhibitor/antagonist; mutant; partition protein parB; promoter; protein function; public health relevance; research study; segregation; small molecule; time use; transcription factor

DESCRIPTION (provided by applicant): Our goal is to define how a bacterial cell integrates the timing of DNA replication and the polar positioning of the chromosomal origin region to control the temporal and spatial expression of cell cycle events. A lynch-pin in the temporal coordination of replication initiation and the transcription of genes encoding global cell cycle regulators is DnaA. This protein functions both to initiate replication and to activate the transcription of over 40 cell cycle-controlled genes. DnaA is controlled at multiple levels, including transcriptional regulation by differential methylation of its promoter that is linked to the progression of the replication fork, and the control of its activity by the replisome-associated HdaA protein. We will determine if changes in DNA methylation state that functions to 'clock' the sequential transcription of both dnaA and ctrA controls the temporal transcription of multiple cell cycle regulated genes, including those controlled by GcrA master regulator. We will also determine if a temporally-controlled dnaA anti- sense transcript contributes to the control of dnaA expression. The dynamic spatial deployment of proteins and the origin region of the chromosome are critical factors in cell cycle control. We have shown that upon replication of the chromosomal origin, the actual DNA sequence that moves toward, and is captured by, the cell pole is parS bound to the ParB partition protein. If the ParB/parS complex is not anchored to the pole, segregation of the rest of the chromosome is impaired and the FtsZ division ring is misplaced. We have recently identified PopZ as a protein that forms a polar polymeric network and functions to anchor ParB/parS to the new cell pole. Important questions are how the polar ribosome free zone formed by the PopZ network is established and how it functions. To define how ParB/parS moves across the cell to be captured by PopZ, we have initiated an analysis of the essential ParA segregation protein. We have generated multiple mutants of ParA and visualized ParA behavior both in vivo and in vitro. We will now identify and characterize the factors that mediate its role in DNA movement and determine the mechanism that drives chromosome segregation. PUBLIC HEALTH RELEVANCE: Based on our elucidation of the genetic circuitry that runs a bacterial cell cycle, we designed a new class of boron-based antibiotics that are in phase two trials. In addition, we identified a small molecule inhibitor of the MreB bacterial actin, with the goal of using this as the basis for a new family of antibiotics.

描述（由申请人提供）：我们的目标是定义细菌细胞如何整合 DNA 复制的时间和染色体起源区域的极性定位来控制细胞周期事件的时间和空间表达。 DnaA 是复制起始和编码全局细胞周期调节基因转录的时间协调的关键。该蛋白质的作用是启动复制并激活 40 多个细胞周期控制基因的转录。 DnaA 在多个层面上受到控制，包括通过其启动子的差异甲基化（与复制叉的进展相关）进行转录调节，以及通过复制体相关的 HdaA 蛋白对其活性进行控制。我们将确定 DNA 甲基化状态的变化是否对 dnaA 和 ctrA 的顺序转录进行“计时”，控制多个细胞周期调节基因的时间转录，包括由 GcrA 主调节器控制的基因。我们还将确定时间控制的 dnaA 反义转录物是否有助于控制 dnaA 表达。蛋白质的动态空间部署和染色体的起源区域是细胞周期控制的关键因素。我们已经证明，在染色体起源复制时，向细胞极移动并被细胞极捕获的实际 DNA 序列是 parS 与 ParB 分区蛋白结合的。如果 ParB/parS 复合体未锚定在极上，则染色体其余部分的分离会受到损害，并且 FtsZ 分裂环会错位。我们最近发现 PopZ 是一种形成极性聚合物网络的蛋白质，其功能是将 ParB/parS 锚定到新的细胞极。重要的问题是 PopZ 网络形成的极性核糖体自由区是如何建立的以及它如何发挥作用。为了确定 ParB/parS 如何跨细胞移动以被 PopZ 捕获，我们开始对必需的 ParA 分离蛋白进行分析。我们已经生成了 ParA 的多个突变体，并在体内和体外观察了 ParA 的行为。我们现在将识别和表征介导其在 DNA 运动中的作用的因素，并确定驱动染色体分离的机制。 公共健康相关性：根据我们对运行细菌细胞周期的遗传电路的阐明，我们设计了一种新型硼基抗生素，目前正在进行第二阶段试验。此外，我们还发现了一种 MreB 细菌肌动蛋白的小分子抑制剂，目标是将其作为新抗生素家族的基础。