Early steps of Bacillus subtilis primosome assembly
Early steps of Bacillus subtilis primosome assembly
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DOI：
10.1074/jbc.m101996200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
Polard, P
中科院分区：
文献类型：
--
作者：
Marsin, S;McGovern, S;Polard, P
引发体是核蛋白组装体，设计用于激活 DNA 复制叉。它们的主要作用是将复制解旋酶募集到单链 DNA 上。大肠杆菌中定义的“复制重启”引发体参与停滞的复制叉的重新激活。 PriA 蛋白与分叉 DNA 的结合会触发其组装。 PriA 在细菌中是保守的，但它的原始伙伴却不是。在枯草芽孢杆菌中，遗传分析揭示了三种初级体蛋白：DnaB、DnaD 和 DnaI，它们在大肠杆菌中没有明显的同源物。有趣的是，它们在复制叉停滞和染色体起源处都参与引发体功能。我们对 DnaB 和 DnaD 蛋白的生化分析揭示了它们在引发体组装中的作用。它们都是多聚体并单独与 DNA 结合。此外，DnaD 刺激 DnaB 结合活性。单独的 DnaD 和 DnaD/DnaB 对在几种 DNA 底物上与枯草芽孢杆菌的 PriA 特异性相互作用。这表明核蛋白组装是按 PriA、DnaD、DnaB 顺序进行的。优选的DNA底物模拟具有未复制的滞后链的停滞的DNA复制叉，其结构与停滞的复制叉的重组修复产物相同。
Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB, and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.