Study on interaction and intracellular distribution of proteins regulating the cell cycle progression in Bacillus subtilis

(1)染色体复制的起始始于启动蛋白dna和oriC序列复合物的形成。然后，DNA解旋酶（DnaC）通过DnaB、dnaad和DnaI蛋白的作用被装载到复合体上。我们发现DnaI作为解旋酶装载器，并与dna相互作用。(2) DnaA分布在细胞质中，但在复制开始时，DnaB和DnaI似乎都与oriC一起定位在类核的外边缘或内缘附近。此外，在复制开始时，在类核的两侧边缘附近检测到DnaX （DNA聚合酶的一种成分）灶，这表明在枯草芽孢杆菌中，复制体被招募到类核两侧边缘附近的oriC中，以启动染色体复制。(3)我们得到的结果表明，如果dna水平随着复制周期的进展而增加，它可能是枯草芽孢杆菌复制起始的限速因子。我们发现，spoj对染色体oriC区域的定位有重要作用。此外，在野生型细胞中，spoOJ突变体产生的染色体含量增加的细胞占很大比例。此外，我们证明了Soj干扰对复制起始的严格控制，导致早期和异步起始，而spoOJ抵消了这种Soj功能。此外，我们发现必需的gtp结合蛋白Bex和YqeH似乎参与了染色体复制起始的调控。(4)我们证明了dnaA基因上和下游的两个DNA序列参与细胞内复制起始区域的正确定位，这一功能与枯草芽孢杆菌染色体的准确分离有关。(5)同时观察时，SMC和复制体通常位于细胞的相似区域。SMC病灶的持续存在并不依赖于正在进行的复制，而是依赖于与SMC相互作用的两种蛋白ScpA和ScpB。我们的研究结果表明，SMC与染色体上的许多位点结合，SMC的浓度定位在复制叉附近，可能在那里结合和组织新复制的DNA。少

(1) Initiation of chromosome replication starts with formation of a complex of initiator protein DnaA and the oriC sequence. Then, DNA helicase, DnaC, is loaded on the complex by the action of DnaB, DnaD and DnaI proteins. We showed that DnaI acts as a helicase loader and DnaD interact with DnaA.(2) DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication, together with oriC. Furthermore, DnaX (a component of DNA polymerase) foci were detected near either of the edges of the nucleoids at the onset of replication, suggesting that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B.subtilis.(3) We obtained results indicating that, if the DnaA level increases with progression of the replication cycle, it could act as a rate-limiting factor of replication initiation in B.subtilis. We found that SpoOJ contributes to the po … More sitioning of the chromosomal oriC region. In addition, the spoOJ mutant produced a significant proportion of cells with increased chromosome content Furthermore we demonstrated that Soj interferes with tight control of replication initiation and causes early and asynchronous initiation and SpoOJ counteracts this Soj function in wild-type cells. Additionally we found that essential GTP-binding proteins, Bex and YqeH, appeared to participate in the regulation of initiation of chromosome replication.(4) We demonstrated that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B.subtilis.(5) When visualized simultaneously, SMC and the replisome were often in similar regions of the cell. Persistence of SMC foci did not depend on ongoing replication, but did depend on ScpA and ScpB, two proteins interacting with SMC. Our results indicate that SMC is bound to many sites on the chromosome and a concentration of SMC is localized near replication forks, perhaps there to bind and organize newly replicated DNA. Less