McMacken R, Ueda K, Kornberg A
Proc Natl Acad Sci U S A. 1977 Oct;74(10):4190-4. doi: 10.1073/pnas.74.10.4190.
The first step in conversion of varphiX174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a varphiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.
在体外将φX174单链DNA转化为双链复制形式的第一步是合成一种核蛋白中间体[韦纳，J. H.，麦克马肯，R. & 科恩伯格，A.（1976年）《美国国家科学院院刊》73，752 - 756]。我们现在证明，dnaB蛋白（每个DNA环约一个分子）是该中间体的必需成分，并保留其ATP酶活性。依赖于dnaG蛋白（引发酶）的RNA引物合成仅发生在已转化为中间体形式的DNA上。在一个耦合的RNA引发 - DNA复制反应中，合成的第一个引物被DNA聚合酶III全酶延伸为全长互补链DNA。在与复制解偶联的RNA引发过程中，在一个φX174环上起始了多个RNA引物。每个DNA环上存在的单个dnaB蛋白分子参与了每个RNA引物的起始，这些引物似乎沿着模板链以规则间隔排列。我们提出，一旦与模板结合，dnaB蛋白以一种连续的方式沿着DNA链迁移，可能利用ATP水解释放的能量进行推进；在这个模型中，活跃移动的dnaB蛋白作为dnaG蛋白（引发酶）产生许多RNA引物的“移动启动子”信号。提出了关于dnaB蛋白参与大肠杆菌染色体复制起点处复制起始以及复制叉处新生（冈崎）片段引物起始的模型。
