ROLE OF TOPOISOMERASES IN DNA REPLICATION

闭环双链质粒DNA的复制， pBR 322已用纯化的蛋白质重构。 复溶 系统包含：RNA聚合酶全酶、核糖核酸酶H、DNA聚合酶 I，DNA聚合酶III全酶，单链DNA结合蛋白， dnaB、dnaC和dnaG（引发酶）蛋白，蛋白i、n、n'（复制 因子Y）和n”，DNA促旋酶和拓扑异构酶I。 复制继续 根据基于先前研究的明确标准， 体内和体外对Co1E1和pBR 322 DNA复制的影响， I型DNA产物。 最近的主要发现是：i）DNA促旋酶提供了 复制叉前进的驱动力（已知的DNA 不需要解旋酶），ii）拓扑异构酶I赋予解旋酶特异性， 复制反应（即，限制DNA模板的起始 含有pBR322型复制起点）和iii）还需要 以影响子DNA分子的分离。 一个复制系统的存在， 纯化的蛋白质提供了操纵系统的能力 生物化学和协调许多部分反应和子系统， 让我们可以研究单个蛋白质的作用， 总结，使整体。 建议的研究将详细说明，首先， 时间，超螺旋上DNA复制的一些基本机制 DNA包括i）连接数的微小变化（由 DNA的拓扑异构酶I）控制DNA复制的起始，ii）DNA的拓扑异构酶I）控制DNA复制的起始。 参与子双链体分离的酶机制， iii）子分子的分离途径， 单独的机制，以及耦合的合成的方式， 领先和落后股和v）负责机械 在DNA复制过程中解旋亲本双链体， 拓扑约束

The replication of the closed-circular, double-stranded plasmid DNA, pBR322, has been reconstituted with purified proteins. The reconstituted system contains: RNA polymerase holoenzyme, ribonuclease H, DNA polymerase I, DNA polymerase III holoenzyme, the single-stranded DNA binding protein, the dnaB, dnaC and dnaG (primase) proteins, proteins i, n, n' (replication factor Y), and n", DNA gyrase and topoisomerase I. Replication proceeds faithfully, according to well-defined criteria based on previous studies in vivo and in vitro on the replication of Co1E1 and pBR322 DNAs, yielding form I DNA product. Major recent findings are that i) DNA gyrase provides the driving force for advance of the replication fork (the known DNA helicases are not required), ii) topoisomerase I imparts specificity to the replication reaction (i.e., restricts initiation to DNA templates containing a pBR322-type origin of replication) and iii) is also required to effect segregation of the daughter DNA molecules. The existence of a replication system completely reconstituted with purified proteins provides the ability to manipulate the system biochemically and orchestrate many partial reactions and subsystems that allow one to study the roles of individual proteins and how these parts sum-up to make the whole. The studies proposed will detail, for the first time, some of the fundamental mechanisms of DNA replication on superhelical DNAs including i) how slight changes in the linking number (induced by topoisomerase I) of the DNA governs initiation of DNA replication, ii) the enzymatic mechanisms involved in the segregation of the daughter duplexes, iii) the pathway of segregation of the daughter molecules, iv) the individual mechanisms for, and the manner of coupling of the synthesis of the leading- and lagging-strand and v) the machinery responsible for unwinding the parental duplex during DNA replication that is under topological constraint.