Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication
Functional interplay between the Bacillus subtilis DnaD and DnaB proteins essential for initiation and re-initiation of DNA replication
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DOI：
10.1111/j.1365-2958.2004.04451.x
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发表时间：
2005-02-01
影响因子：
3.6
通讯作者：
Polard, P
中科院分区：
文献类型：
--
作者：
Bruand, C;Velten, M;Polard, P
细菌中染色体DNA复制的起始和再起始依赖于趋异的多蛋白组装体，其指导单链DNA（ssDNA）上的复制解旋酶在特定位点的功能递送。这两个过程分别由保守的DnaA和PriA蛋白在单个染色体起点oriC或被捕叉处触发。在枯草芽孢杆菌中，这两种途径还需要在低GC含量的革兰氏阳性细菌中限制性编码的三种必需蛋白DnaB、DnaD和DnaI。我们最近表明，DnaI和DnaB作为一对装载的DnaC复制解旋酶。DnaD的作用似乎更加神秘。它以前被证明与DnaA相互作用，并显示弱ssDNA结合活性。在这里，我们报告说，纯化的DnaD可以与PriA和DnaB的物理相互作用。我们发现，温度敏感的dnaD23突变体的致死率可以抑制不同的DnaB点突变体，这被发现是相同的抑制蛋白A无效突变体。DnaD23蛋白显示出比DnaD更低的ssDNA结合活性。相反，主要的dnaD23抑制因子DnaB75蛋白获得了对ssDNA的亲和力。最后，我们观察到，DnaD和DnaB之间的这种相互作用是至关重要的，它们与SSB包被的ssDNA，这是预期的基板在体内的复制解旋酶的负载协调相互作用。总而言之，这些结果突出了DnaD和DnaB在染色体DNA复制的起始和再起始的早期阶段单独和一起与ssDNA相互作用的需要。他们还指出了DnaD在这两个基本过程中构建的多蛋白组装中的主要结构作用。
Initiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis, these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSB-coated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo. Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.