DEVELOPMENT CONTROL OF DNA REPLICATION IN CAULOBACTER

我们的目标是了解控制时间的机制 DNA复制的启动是细胞周期的函数。我们建议 为了研究新月弯杆菌的这一关键事件，新月弯杆菌是一种其 发育细胞周期表现出固有的不对称性。最新的 复制的染色体具有不同的复制潜力：染色体 分裂到子代有柄的细胞立即启动DNA 复制，而后代蜂群细胞染色体不启动 复制直到细胞周期的后期。硫杆菌是唯一适合的细菌 对控制DNA复制的因素的研究，因为它有一个 从已确定的起源开始复制的单条染色体 每个细胞周期一次，细胞类型可以很容易地从 同步的种群。我们最近分离出了微型染色体 仅由正确启动复制的克隆源驱动 与真正的染色体起源相吻合。拟议的研究 有四个主要目标。第一种方法是定义复制源 介导差异启动的序列和同源因素 DNA复制。为此，我们将在体内和体外鉴定， 与起源区域相结合的因子，这些区域对 复制启动或复制的时间。我们还将 确定启动子是否驻留在原点内并且 从复制能力强的染色体中选择性表达，有助于 涉及复制启动的控制。我们还将尝试直接 在分区前的一极可视化原产地特定的成绩单 细胞进行原位杂交。第二个目标是隔离和 DNA复制和/或染色体缺陷突变体的特征 种族隔离。三是确定控制经济增长的机制 已鉴定的酶及其相关因素的细胞周期表达 在DNA复制中，例如DNA聚合酶III的β亚基DNAA， 和旋转酶。第四个目标是确定 调控一种新发现的DNA的细胞周期表达 甲基转移酶，并研究这一甲基化系统在 复制启动和细胞分化的控制。这些研究 将解决控制极性的基本机制 细菌细胞周期。

Our goal is to understand the mechanisms that control the timing of the initiation of DNA replication as a function of the cell cycle. We propose to study this crucial event in Caulobacter crescentus, an organism whose developmental cell cycle exhibits inherent asymmetry. The newly replicated chromosomes differ in replication potential: the chromosome that partitions to the progeny stalked cell immediately initiates DNA replication whereas the progeny swarmer cell chromosome does not initiate replication until later in the cell cycle. Caulobacter is uniquely suited to a study of the factors that control DNA replication because it has a single chromosome that initiates replication from an identified origin once per cell cycle, in a cell type that can be easily obtained from synchronized populations. We have recently isolated mini-chromosomes driven solely by the cloned origin that correctly initiate replication coincident with the bona fide chromosomal origin. The proposed research has four main objectives. The first is to define the replication origin sequences and cognate factors that mediate the differential initiation of DNA replication. To do this we will identify, both in vivo and in vitro, factors that bind to regions of the origin that are essential for replication initiation or for the timing of replication. We will also determine if a promoter, shown to reside within the origin and to be selectively expressed from replication-competent chromosome, contributes to the control of replication initiation. We will also attempt to directly visualize origin-specific transcripts at one pole of the predivisional cell using in situ hybridization. The second objective is to isolate and characterize mutants defective in DNA replication and/or chromosome segregation. The third is to determine the mechanisms that control the cell cycle expression of identified enzymes and factors that are involved in DNA replication, such as DnaA, the beta subunit of DNA polymerase III, and gyrase. The fourth objective is to determine the mechanisms that regulate the cell cycle expression of a newly identified DNA methyltransferase, and examine the role of this methylation system in the control of replication initiation and cell differentiation. These studies will address the underlying mechanisms that control polarity during the bacterial cell cycle.