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

枯草芽孢杆菌细胞周期进程蛋白质相互作用及胞内分布研究

• 批准号: 11480212
• 负责人: OGASAWARA Naotake
• 金额: $ 8.13万
• 依托单位: NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480212/
• 关键词: Bacillus subtilis; Chromosome replication; Chromosome segregation; DnaA protein; Initiation complex; Replisome; SpoOJ protein; SMC protein; 染色体構造; Spo0J蛋白質; Smc蛋白質; 複製開始; DnaA; 細胞周期; フローサイトメトリー; 染色体分離; シス配列; FISH; 蛋白質相互作用; 酵母2ハイブリッド系

(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

(1)染色体复制的起始起始于起始蛋白DnaA和oriC序列的复合物的形成。然后，DNA解旋酶DnaC通过DnaB、DnaD和DnaI蛋白质的作用加载到复合物上。我们发现，DnaI作为一个解旋酶加载和DnaD与DnaA相互作用。(2)DnaA分布在整个细胞质中，但DnaB和DnaI似乎都定位在复制起始时的类核的外边缘或内边缘附近，以及oriC。此外，DnaX（DNA聚合酶的一个组成部分）焦点附近的任一边缘的类核在复制开始时检测到，这表明复制体被招募到oriC附近的任一边缘的类核启动染色体复制在枯草杆菌。(3)我们得到的结果表明，如果DnaA水平随着复制周期的进展而增加，它可以作为一个限速因子的复制启动在枯草杆菌。我们发现SpoOJ有助于PO ...更多信息 染色体oriC区域的定位。此外，spoOJ突变体产生了显着比例的细胞染色体含量增加。此外，我们证明，Soj干扰复制启动的严格控制，并导致早期和异步启动和SpoOJ抵消这种Soj功能在野生型细胞。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节。(4)我们证明了这两个DNA序列中和下游的dnaA基因参与正确定位的复制起点区域内的细胞，这种功能与准确的染色体分离在枯草杆菌。(5)当同时观察时，SMC和复制体通常位于细胞的相似区域。SMC病灶的持久性不依赖于正在进行的复制，但确实依赖于ScpA和ScpB，两种蛋白与SMC相互作用。我们的研究结果表明，SMC是绑定到染色体上的许多网站和SMC的浓度是本地化的复制叉附近，也许有绑定和组织新复制的DNA。少

项目成果

Soppa, J., Kobayashi, K., Noirot-Gros, M.F., Oesterhelt, D., Ehrlich, S.D., Dervyn, E., Ogasawara, N., and Moriya, S.: "Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Ba

Soppa, J.、Kobayashi, K.、Noirot-Gros, M.F.、Oesterhelt, D.、Ehrlich, S.D.、Dervyn, E.、Ogasawara, N. 和 Moriya, S.：“发现了两个新的蛋白质家族，

Lee, P.S., Lin, D.C., Moriya, S., and Grossman, A.D.: "Effects of the chromosome partitioning protein SpoOJ (ParB) on oriC positioning and replication initiation in Bacillus subtilis"J Bacteriol. 185. 1326-1337 (2003)

Lee, P.S.、Lin, D.C.、Moriya, S. 和 Grossman, A.D.：“染色体分配蛋白 SpoOJ (ParB) 对枯草芽孢杆菌中 oriC 定位和复制起始的影响”J Bacteriol。

Ogura, Y.: "Autoregulation of the dnaA-dnaN operon and effects of DnaA protein levels on replication initiation in Bacillus subtilis"J. Bacteriol. 183・6. 3833-3841 (2001)

Ogura, Y.：“枯草芽孢杆菌中 dnaA-dnaN 操纵子的自动调节和 DnaA 蛋白水平对复制起始的影响”J. Bacteriol. 183・6 3833-3841 (2001)。

Lemon, K.P.: "Bacillus subtilis and Its Closest Relatives : from Genes to Cells"ASM Press. 629 (2002)

Lemon, K.P.：“枯草芽孢杆菌及其近亲：从基因到细胞”ASM Press。

Imai, Y.: "Subcellular Iocalization of Dna-initiation proteins of Bacillus subtilis : evidence that chromosome replication begins at either edge of the nucleoids"Mol Microbiol. 36. 1037-1048 (2000)

Imai, Y.：“枯草芽孢杆菌 DNA 起始蛋白的亚细胞定位：染色体复制开始于核仁任一边缘的证据”Mol Microbiol。