Leonard Alan C, Grimwade Julia E
EcoSal Plus. 2010 Sep;4(1). doi: 10.1128/ecosalplus.4.4.1.
In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC, the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli, in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC, were identified. The DnaA protein is the bacterial initiator, and in E. coli, the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC, the ordered assembly and disassembly of a multi-DnaA complex at oriC, and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.
近年来已经明确，复杂的调控回路通过指导多组分分子机器（复制起始复合物）的组装来控制DNA复制的起始步骤，该分子机器在oriC（唯一的染色体复制起点）处分离DNA链并加载复制性解旋酶。本章讨论了最近为理解负责染色体复制适时起始的调控性蛋白质-DNA相互作用所做的努力。它回顾了有关大肠杆菌oriC内新鉴定的核苷酸序列特征以及细菌起始蛋白DnaA的新结构和生化特性的信息。它还讨论了防止DNA复制时间不当的协调机制。编码起始蛋白的基因的鉴定来自对大肠杆菌温度敏感、条件致死突变体的研究，在这些突变体中鉴定出了两种DNA复制缺陷表型，即“立即停止”突变体和“延迟停止”突变体。延迟停止突变体的动力学表明，缺陷基因产物是DNA合成起始步骤所特需的，随后，鉴定出了两个基因，即dnaA和dnaC。DnaA蛋白是细菌起始蛋白，在大肠杆菌中，需要DnaC蛋白来加载复制性解旋酶。DnaA对oriC的可及性调控、oriC处多DnaA复合物的有序组装和解聚以及DnaA解开oriC的方式仍然是有待回答的重要问题，本章讨论了这些主题的当前知识状态。
