Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading.
Overexpression of the Replicative Helicase in Escherichia coli Inhibits Replication Initiation and Replication Fork Reloading.
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DOI：
10.1016/j.jmb.2016.01.018
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发表时间：
2016-03-27
影响因子：
5.6
通讯作者：
McGlynn P
中科院分区：
文献类型：
--
作者：
Brüning JG;Myka KK;McGlynn P
复制解旋酶在染色体复制中发挥核心作用，它们在 DNA 上的组装通过启动子和解旋酶装载蛋白进行调节。大肠杆菌复制解旋酶 DnaB 和解旋酶装载机 DnaC 形成 DnaB6-DnaC6 复合物，该复合物是将 DnaB 装载到单链 DNA 上所必需的。 dnaC 的过度表达通过促进 DnaC 与 DnaB 的持续重新结合以及随后防止解旋酶易位来抑制复制。在这里，我们发现 dnaB 的过度表达也会抑制生长和染色体复制。这种抑制作用被野生型 DnaC 的共过表达所抵消，但不能与 DnaB 相互作用的 DnaC 突变体的共过表达抵消，表明 DnaB6–DnaC6 浓度的降低是与 DnaB 浓度升高相关的表型的原因。 oriC 特异性起始子 DnaA 和 PriA 特异性起始在复制修复过程中远离 oriC 的部分缺陷使细胞对 dnaB 过度表达敏感。辅助复制解旋酶 Rep 的缺失会导致复制阻断增加，从而增加远离 oriC 的重新启动，也会加剧 DnaB 诱导的缺陷。这些发现表明，解旋酶水平升高不仅会扰乱复制起点的复制起始，还会扰乱染色体其他位点的分叉修复过程。因此，复制解旋酶和解旋酶装载机水平的不平衡可以通过抑制过量 DnaB 形成 DnaB6-DnaC6 复合物（如此处所示）和促进过量 DnaC 形成 DnaB6-DnaC6 复合物来抑制复制 [Allen GC, Jr., Kornberg A. 大肠杆菌复制中 DnaC 蛋白调节 DnaB 解旋酶的精细平衡。 J.Biol。化学。 1991;266:22096–22101; Skarstad K，Wold S。体内大肠杆菌分叉的速度取决于 DnaB：DnaC 比率。摩尔。微生物。 1995；17：825–831]。因此，复制解旋酶及其相关装载蛋白的不平衡可以通过两种机制抑制基因组复制。复制解旋酶的加载是复制体组装的关键步骤。增加大肠杆菌中的复制解旋酶浓度会抑制生长。抑制是由于解旋酶复合物耗尽了解旋酶装载蛋白。耗尽会抑制复制修复过程中的复制启动和重新启动。复制解旋酶组分的不平衡会阻止复制启动。
Replicative helicases play central roles in chromosome duplication and their assembly onto DNA is regulated via initiators and helicase loader proteins. The Escherichia coli replicative helicase DnaB and the helicase loader DnaC form a DnaB6–DnaC6 complex that is required for loading DnaB onto single-stranded DNA. Overexpression of dnaC inhibits replication by promoting continual rebinding of DnaC to DnaB and consequent prevention of helicase translocation. Here we show that overexpression of dnaB also inhibits growth and chromosome duplication. This inhibition is countered by co-overexpression of wild-type DnaC but not of a DnaC mutant that cannot interact with DnaB, indicating that a reduction in DnaB6–DnaC6 concentration is responsible for the phenotypes associated with elevated DnaB concentration. Partial defects in the oriC-specific initiator DnaA and in PriA-specific initiation away from oriC during replication repair sensitise cells to dnaB overexpression. Absence of the accessory replicative helicase Rep, resulting in increased replication blockage and thus increased reinitiation away from oriC, also exacerbates DnaB-induced defects. These findings indicate that elevated levels of helicase perturb replication initiation not only at origins of replication but also during fork repair at other sites on the chromosome. Thus, imbalances in levels of the replicative helicase and helicase loader can inhibit replication both via inhibition of DnaB6–DnaC6 complex formation with excess DnaB, as shown here, and promotion of formation of DnaB6–DnaC6 complexes with excess DnaC [Allen GC, Jr., Kornberg A. Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli. J. Biol. Chem. 1991;266:22096–22101; Skarstad K, Wold S. The speed of the Escherichia coli fork in vivo depends on the DnaB:DnaC ratio. Mol. Microbiol. 1995;17:825–831]. Thus, there are two mechanisms by which an imbalance in the replicative helicase and its associated loader protein can inhibit genome duplication. Loading of the replicative helicase is the key step in replisome assembly. Increasing replicative helicase concentration in E. coli inhibits growth. Inhibition is due to helicase complexes depleted of the helicase loader protein. Depletion inhibits replication initiation and reinitiation during replication repair. Imbalances in replicative helicase components can prevent replication initiation.