Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex -: art. no. 28
Dynamic assembly, localization and proteolysis of the Bacillus subtilis SMC complex -: art. no. 28
复制标题
DOI：
10.1186/1471-2121-6-28
复制
发表时间：
2005-06-29
期刊：
影响因子：
--
通讯作者：
Graumann, PL
中科院分区：
文献类型：
--
作者：
Mascarenhas, J;Volkov, AV;Graumann, PL
背景：SMC蛋白是几种蛋白质复合物的关键组分，在不同的染色体动力学中执行重要任务。细菌SMC与ScpA和ScpB形成复合物，对染色体排列和分离至关重要。复合体定位于在细胞周期的大部分时间内以双极方式定位的类核上的离散中心。这种复合物以一种未知的方式与DNA结合并凝结DNA。结果：我们发现ScpA和ScpB在体外形成不同的复合物，其中假定的2 ScpA/4 ScpB复合物的水平在添加SMC蛋白后显着降低。SMC的atpase结合袋的不同突变降低了SMC与ScpA和ScpB的相互作用，但没有消除突变SMC与ScpA和ScpB的相互作用。SMC atp酶活性的丧失导致体内功能的丧失，并取消SMC复合物的适当定位。在抑制gyrase活性后，双极性SMC中心的形成也丢失，并且在抑制复制期间异常，导致单中心簇。复制的恢复迅速重新建立了双极SMC中心，表明适当的定位取决于正在进行的复制。我们还发现SMC蛋白受到诱导蛋白水解，最显著的是当细胞进入静止期时，这部分是由ClpX和LonA蛋白酶实现的。原子力显微镜显示体外高阶玫瑰花状SMC结构的存在，这可能解释了体内SMC中心的形成。结论：我们的数据表明ScpA/ScpB亚复合物被直接招募到SMC复合物中。这个过程不需要SMC atp酶活性，然而，它似乎有助于ScpA和ScpB的加载。因此，SMC的活性可以通过ScpA和ScpB的结合和释放来调节，而ScpA和ScpB已被证明会影响SMC的atp酶活性。SMC复合体的正确双极定位取决于多种生理方面：正在进行的复制，atp酶活性和染色体超盘绕。由于SMC蛋白的细胞浓度也在转录后水平受到调控，因此SMC的活性显然在多个水平上受到调控。
Background: SMC proteins are key components of several protein complexes that perform vital tasks in different chromosome dynamics. Bacterial SMC forms a complex with ScpA and ScpB that is essential for chromosome arrangement and segregation. The complex localizes to discrete centres on the nucleoids that during most of the time of the cell cycle localize in a bipolar manner. The complex binds to DNA and condenses DNA in an as yet unknown manner.Results: We show that in vitro, ScpA and ScpB form different complexes with each other, among which the level of the putative 2 ScpA/4 ScpB complex showed a pronounced decrease in level upon addition of SMC protein. Different mutations of the ATPase-binding pocket of SMC reduced, but did not abolish interaction of mutant SMC with ScpA and ScpB. The loss of SMC ATPase activity led to a loss of function in vivo, and abolished proper localization of the SMC complex. The formation of bipolar SMC centres was also lost after repression of gyrase activity, and was abnormal during inhibition of replication, resulting in single central clusters. Resumption of replication quickly re-established bipolar SMC centres, showing that proper localization depends on ongoing replication. We also found that the SMC protein is subject to induced proteolysis, most strikingly as cells enter stationary phase, which is partly achieved by ClpX and LonA proteases. Atomic force microscopy revealed the existence of high order rosette-like SMC structures in vitro, which might explain the formation of the SMC centres in vivo.Conclusion: Our data suggest that a ScpA/ScpB sub-complex is directly recruited into the SMC complex. This process does not require SMC ATPase activity, which, however, appears to facilitate loading of ScpA and ScpB. Thus, the activity of SMC could be regulated through binding and release of ScpA and ScpB, which has been shown to affect SMC ATPase activity. The proper bipolar localization of the SMC complex depends on a variety of physiological aspects: ongoing replication, ATPase activity and chromosome supercoiling. Because the cellular concentration of SMC protein is also regulated at the posttranscriptional level, the activity of SMC is apparently regulated at multiple levels.