Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.
Regulation of the activity of the dual-function DnaA protein in Caulobacter crescentus.
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DOI：
10.1371/journal.pone.0026028
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Collier J
中科院分区：
文献类型：
--
作者：
Fernandez-Fernandez C;Gonzalez D;Collier J
DNAA是一种保守的必需细菌蛋白，在新月形杆菌中既是染色体复制的起始者，又是主要的转录调节因子。因此，细胞内活性DNAA的水平需要在细胞周期中受到严格的调控。我们以前的工作表明，DNAA可能受复制体相关蛋白HdaA在其活性水平的调节。在这里，我们描述了突变的Dna A蛋白[Dna A(R357A)]的构建。Crescentus Dna A蛋白AAA+结构域的R357残基相当于大肠杆菌Dna A蛋白的R334残基，这是Dna(RIDA)调节失活所必需的。我们发现，在crescentus中表达Dna A(R357A)突变蛋白，而不是表达类似水平的野生型Dna A蛋白，会导致严重的过度启动染色体复制的表型，并阻止细胞分裂。因此，与野生型Dna A蛋白相比，突变的Dna A(R357A)蛋白在促进DNA复制的启动方面具有高度的活性。在体内，Dna A(R357A)不能取代Dna A，这表明一旦染色体复制开始，Dna A活性的转换可能是新月菱形藻的一个重要过程。我们认为，DNAA的失活是确保每个细胞周期只开始一次染色体复制的主要机制。我们进一步观察到，Dna A中的R357A替换并没有促进Dna A作为四个重要基因的直接转录激活因子的活性，这四个基因编码HdaA、GcrA主细胞周期调节因子、FtsZ细胞分裂蛋白和细胞分裂的MipZ空间调节因子。因此，DNAA的AAA+结构域可能通过将DNAA分子从启动DNA复制重新分配到转录基因，从而在时间上调节DNAA的双功能。
DnaA is a conserved essential bacterial protein that acts as the initiator of chromosomal replication as well as a master transcriptional regulator in Caulobacter crescentus. Thus, the intracellular levels of active DnaA need to be tightly regulated during the cell cycle. Our previous work suggested that DnaA may be regulated at the level of its activity by the replisome-associated protein HdaA. Here, we describe the construction of a mutant DnaA protein [DnaA(R357A)]. The R357 residue in the AAA+ domain of the C. crescentus DnaA protein is equivalent to the R334 residue of the E. coli DnaA protein, which is required for the Regulatory Inactivation of DnaA (RIDA). We found that the expression of the DnaA(R357A) mutant protein in C. crescentus, but not the expression of the wild-type DnaA protein at similar levels, causes a severe phenotype of over-initiation of chromosomal replication and that it blocks cell division. Thus, the mutant DnaA(R357A) protein is hyper-active to promote the initiation of DNA replication, compared to the wild-type DnaA protein. DnaA(R357A) could not replace DnaA in vivo, indicating that the switch in DnaA activity once chromosomal replication has started may be an essential process in C. crescentus. We propose that the inactivation of DnaA is the main mechanism ensuring that chromosomal replication starts only once per cell cycle. We further observed that the R357A substitution in DnaA does not promote the activity of DnaA as a direct transcriptional activator of four important genes, encoding HdaA, the GcrA master cell cycle regulator, the FtsZ cell division protein and the MipZ spatial regulator of cell division. Thus, the AAA+ domain of DnaA may play a role in temporally regulating the bifunctionality of DnaA by reallocating DnaA molecules from initiating DNA replication to transcribing genes within the unique DnaA regulon of C. crescentus.
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