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

(1)染色体复制起始于启动子蛋白DNAA和ORIC序列的复合体的形成。然后，在DNAB、DNAD和DNAi蛋白的作用下，DNA解旋酶DNAC被负载到复合体上。我们发现DNAi作为解旋酶的载入器，DNAD与DNAA相互作用。(2)DNAA分布在细胞质中，但在复制开始时，DNAB和DNAi似乎都定位在类核的外缘或内缘附近，与ORIC一起。此外，在复制开始时，在类核的任何一个边缘附近都检测到了DNAX(DNA聚合酶的一种成分)焦点，这表明在枯草杆菌中，复制体被招募到靠近类核的边缘的ORIC中，以启动染色体复制。(3)我们的结果表明，如果Dna A水平随着复制周期的进展而增加，它可能是枯草杆菌复制启动的一个限速因子。我们发现spoj对po…有贡献。更多的染色体ORIC区域大小。此外，SpoOJ突变体产生了相当大比例的细胞染色体含量增加。此外，我们还证明了SOJ干扰复制启动的严格控制，并导致早期和异步启动，SpoOJ在野生型细胞中抵消了这一SOJ功能。此外，我们还发现必需的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Dna A基因及其下游的两个DNA序列参与了细胞内复制起始区的正确定位，这一功能与枯草杆菌染色体的准确分离有关。(5)当同时观察到SMC和复制体时，SMC和复制体通常位于细胞的相似区域。SMC病灶的持久性不依赖于正在进行的复制，而取决于与SMC相互作用的两种蛋白质SCPA和SCPB。我们的结果表明，SMC与染色体上的许多位置结合，SMC集中在复制叉附近，可能是为了结合和组织新复制的DNA。较少

(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