INITIATION OF DNA REPLICATION IN THE PHAGE T4 SYSTEM

描述(申请人提供)：噬菌体T4中的DNA复制启动 通过两种不同的模式，这两种模式都需要特定的DNA结构。起源 复制从R环开始，而依赖重组的复制 (RDR)从D-环路启动。将使用拓扑分析来测量 T4的R-环形成起源于体内。结果应该会澄清 R环的形成机制及UvsW蛋白解旋模型的验证 在后期抑制T4起源的R-环。复制的详细角色 蛋白质和原始核糖核酸也将被分析，例如询问 源RNA的翻译减少了复制，决定了GP59 在体内充当复制装置的“分子看门人”，以及 检测T4拓扑异构酶是否是主要的分叉转位酶和十烯酸酶。一种新的 调查区域将尝试隔离活动的复制源 在未感染的大肠杆菌中构成稳定的DNA复制，这也是 建议通过R环机制运行。对T4 RDR的研究揭示了一种 复制、重组和DSB修复之间的紧密联系。产品 DSB修复的需求将在质粒和噬菌体中进行分析 染色体来区分三种不同的模型。此外, DSB促进的DNA复制将在噬菌体的上下文中进行分析 基因组。使用仅在受损区域活跃的噬菌体T4重组热点 DNA，复制叉在损坏和重新启动时阻塞的过程将 也被接洽了。分叉重启可能是RDR的一条特殊途径。这个 拟议的研究具有显著的健康相关性，因为T4系统 继续提供与人类细胞复制相关的重要经验， 重组和修复。这些过程在许多医学上都是至关重要的 相关领域，如早期发育、抗体多样性的产生、 对致癌和抗癌药物的反应，并维持 增殖中的肿瘤细胞中的染色体。

DESCRIPTION (provided by applicant): DNA replication in phage T4 is initiated by two distinct modes, both of which require particular DNA structures. Origin replication initiates from R-loops, while recombination-dependent replication (RDR) initiates from D-loops. A topological assay will be used to measure R-loop formation at T4 origins in vivo. The results should clarify the mechanism of R-loop formation and test the model that UvsW protein unwinds R-loops to repress T4 origins at late times. The detailed roles of replication proteins and origin RNA will also be analyzed, for example asking whether translation of the origin RNA reduces replication, determining whether gp59 acts as a "molecular gatekeeper" of the replication apparatus in vivo, and testing whether T4 topoisomerase is the major fork swivel and decatenase. A new area of investigation will attempt the isolation of replication origins active in constitutive stable DNA replication in uninfected E. coli, which is also proposed to operate by an R-loop mechanism. Studies of T4 RDR have uncovered a tight linkage between replication, recombination and dsb repair. The products and requirements for dsb repair will be analyzed in plasmids and in phage chromosomes to distinguish between three different models. In addition, dsb-promoted DNA replication will be analyzed within the context of the phage genome. Using phage T4 recombination hotspots that are only active on damaged DNA, the process of replication fork blockage at damage and fork restart will also be approached. Fork restart is likely a specialized pathway of RDR. The proposed studies have significant health relatedness because the T4 system continues to provide important lessons relevant to human cell replication, recombination and repair. These processes are critical in many medically relevant areas, such as early development, generation of antibody diversity, response to both carcinogenic and anticancer agents, and maintenance of chromosomes in proliferating tumor cells.