Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis
Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis
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DOI：
10.1074/jbc.m308956200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Biswas, SB
中科院分区：
文献类型：
--
作者：
Mitkova, AV;Khopde, SM;Biswas, SB
在大肠杆菌复制叉的滞后链中启动和合成RNA引物需要复制性DnaB解旋酶和DNA引物酶，即DnaG基因产物。此外，这两种复制酶之间的物理相互作用似乎在染色体DNA复制的起始中起作用。在体外，DnaB解旋酶刺激引物酶在单链（ss）寡核苷酸模板上合成引物。早期的研究假设多个引发酶分子与每个DnaB六聚体和单链DNA相互作用。我们已经研究了这一假设，并确定了确切的化学计量的引物DnaB六聚体。我们还证明了DnaB解旋酶的ssDNA结合活性对于引导引发酶至起始三核苷酸和合成11-20个核苷酸长的引物是必需的。虽然这两种酶的结合决定了RNA引物在体外的合成程度和速率，但在E.大肠杆菌，因为它们相互作用的瞬时性质。因此，我们使用化学交联剂稳定该复合物，并通过凝胶过滤对该复合物进行化学计量分析。这使我们能够证明E.大肠杆菌中的引物酶由三个分子与一个DnaB六聚体结合组成。DnaB与引发酶相互作用的荧光各向异性研究（用荧光探针Ru（bipy）标记）（3）和Scatchard分析进一步支持了这一结论。将DnaC蛋白质添加到简单的引发系统中，导致DnaB-DnaC复合物的形成，导致较短引物的合成。因此，DnaB-引物酶复合物与其他复制因子的相互作用可能对于确定体内RNA引物的生理长度和引物合成的总体动力学至关重要。
Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical crosslinker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)(3), and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.