Biochemical Study on the Initiation Complex of the Replication of the Bacillus Subtilisin Chromosome

1. 通过dna基因产物和oriC的连续作用形成独特的蛋白质- dna复合物（initiosome）被认为是细菌染色体复制起始的先决条件。我们之前发现，在进化中广泛分化的细菌物种中，dna蛋白似乎在开始复制中起着核心作用。然而，决定oriC被dna蛋白激活时间的调控机制尚不清楚。本研究旨在揭示起始酶体的组织结构，以了解起始复制的调控机制。我们之前发现，DnaA基因及其结合序列（DnaA-box）的组合在真细菌中广泛保守，并且枯草芽孢杆菌（Bacillus subtilisin）的DnaA蛋白在体外特异性结合DnaA-box。我们现在分离了枯草芽孢杆菌的温度敏感突变体DnaA，并直接表明DnaA蛋白特异性参与染色体复制的起始。此外，我们还证实了含有两个DNA盒子簇的DNA片段在枯草芽孢杆菌中具有同样的活性。突变的dna蛋白在不允许的温度下是不稳定的。在49℃的第一个孵育期，其数量迅速下降，随后又开始升高，可能是由于自身调节的抑制。这一现象表明，dna蛋白的失活引起酶体的破坏，从而增加酶体对蛋白酶作用的敏感性。利用这一现象，我们证明了dna蛋白的数量与细胞的起始电位之间存在线性关系。另一方面，我们发现枯草芽孢杆菌中dna蛋白的人工过量产生对染色体复制起始的特异性抑制。这一结果表明，还有其他限制复制起始的因素，这些因素将与dna蛋白协同工作。为了阐明这些因素，目前正在对与DnaA蛋白形成复合物的蛋白质进行生化分析，并对DnaA突变的抑制突变进行遗传分析。少

1. Formation of a unique protein-DNA complex (initiosome) by a sequential action of DnaA gene products and oriC is thought to be prerequisite for initiation of chromosomal replication in bacteria. We previously found that DnaA protein seems to play a central role in initiation of replication in bacterial species diverged widely in evolution. However, regulatory mechanism which determines the time of activation of oriC by DnaA protein wa not known. The aim of this research was to unveil the organization of initiosome in order to understand the regulatory mechanism of initiation of replication.2. We previously found that combination of the DnaA gene and its binding sequence (DnaA-box) is conserved widely in eubacteria, and that Bacillus subtilisin DnaA protein binds specifically to DnaA-box in vitro. We now isolated a temperature sensitive mutant of the B. subtilisin DnaA, and showed directly that DnaA protein is specifically involved in initiation of chromosomal replication. In addition … More , we demonstrated that a DNA fragment containing two DnaA-box clusters flanking DnaA gene has ars activity in B. subtilisin.3. The mutant DnaA protein was unstable at the unpermissive temperature. Its amount decreased rapidly during the first incubation period at 49 C, and then started to increase at the elevated level probably due to the derepression of autogenous regulation. This phenomena indicated that inactivation of DnaA protein causes the destruction of the initiosome, and then increasing its sensitivity to protease action. Taking advantage of this phenomena, we showed that there is a linear relationship between the amount of DnaA protein and cell's initiation potential.4. On the other hand, we found that the specific inhibition of initiation of chromosomal replication occurs by the artificial overproduction of the DnaA protein in B. subtilisin. This result indicates that there are other limiting factors for initiation of replication, which will work in concert with the DnaA protein. In order to elucidate such factors, biochemical analysis of proteins which forms a complex with the DnaA protein and genetic analysis of suppressor mutations of the DnaA mutation are now in progress. Less