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

1.通过DnaA基因产物和oriC的顺序作用形成独特的蛋白质-DNA复合物（起始体）被认为是细菌中染色体复制起始的先决条件。我们以前发现，DnaA蛋白似乎在细菌物种的复制起始中起着核心作用，这些细菌物种在进化上有很大差异。然而，DnaA蛋白激活oriC的调控机制尚不清楚。本研究的目的是揭示起始体的组织结构，以了解复制起始的调控机制.我们以前发现，DnaA基因及其结合序列（DnaA-box）的组合在真细菌中是广泛保守的，枯草杆菌蛋白酶DnaA蛋白在体外特异性结合DnaA-box。我们现在分离了B的温度敏感突变体。枯草杆菌蛋白酶DnaA，并直接表明DnaA蛋白特异性参与染色体复制的起始。此外 ...更多信息 结果表明，在B中，含有两个DnaA盒簇的DNA片段具有ars活性。枯草杆菌蛋白酶3.突变体DnaA蛋白在不允许的温度下不稳定。在49 ℃的第一个孵育期内，其含量迅速下降，然后开始升高，可能是由于自体调节的去抑制。这种现象表明，DnaA蛋白的失活导致了起始体的破坏，从而增加了其对蛋白酶作用的敏感性。利用这一现象，我们发现DnaA蛋白的量与细胞的启动电位之间存在线性关系.另一方面，我们发现，通过在B中人为过量产生DnaA蛋白，发生了对染色体复制起始的特异性抑制。枯草杆菌蛋白酶。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DnaA蛋白协同作用。为了阐明这些因素，目前正在进行与DnaA蛋白质形成复合物的蛋白质的生化分析和DnaA突变的抑制突变的遗传分析。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