DEVELOPMENT CONTROL OF DNA REPLICATION IN CAULOBACTER

我们的目标是了解控制时间的机制， 启动DNA复制作为细胞周期的功能。我们提出 研究新月柄杆菌中的这一关键事件， 发育细胞周期表现出固有的不对称性。 新 复制的染色体在复制潜力上不同：染色体 分配到后代有柄细胞的DNA 复制，而后代群体细胞染色体不启动 直到细胞周期的后期。柄杆菌是唯一适合 研究控制DNA复制的因素，因为它有一个 从一个确定的起点开始复制的单个染色体 每个细胞周期一次，在可以容易地从 同步种群。我们最近分离出了微型染色体 完全由正确启动复制的克隆起点驱动 与真正的染色体起源一致。拟议研究 有四个主要目标。首先是定义复制起点 序列和同源因子介导的差异启动 DNA复制。为此，我们将在体内和体外鉴定， 与原产地结合的因素， 复制启动或复制的时间。我们还将 确定启动子是否存在于起源内， 选择性地从有复制能力的染色体上表达， 到复制起始的控制。我们还将尝试直接 在分区前的一个极点可视化特定于起源的转录本 细胞原位杂交。第二个目标是隔离和 表征DNA复制和/或染色体缺陷的突变体 种族隔离第三是确定控制 所鉴定的酶和相关因子的细胞周期表达 在DNA复制中，如DNA聚合酶III的β亚基DnaA， 和促旋酶。 第四个目标是确定 调节新鉴定的DNA的细胞周期表达 甲基转移酶，并检查这种甲基化系统在 控制复制起始和细胞分化。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.