Li Yongfang, Dabrazhynetskaya Alena, Youngren Brenda, Austin Stuart
Gene Regulation and Chromosome Biology Laboratory, National Cancer Institute, CCR, NCI-Frederick, Frederick, MD 21702-1201, USA.
Mol Microbiol. 2004 Jul;53(1):93-102. doi: 10.1111/j.1365-2958.2004.04111.x.
The parS centromere-like site promotes active P1 plasmid segregation in the presence of P1 ParA and ParB proteins. At the modest growth rate used here, time-lapse and still photomicroscopy shows that the plasmid copies are clustered as a focus at the Escherichia coli cell centre. Just before cell division, the focus is actively divided and ejects bidirectionally into opposite halves of the dividing cell. In the absence of the wild-type parS binding protein ParB, a focus was formed, but generally did not go to the cell centre. The randomly placed focus did not divide and was inherited by one daughter cell only. In the absence of ParA, foci formed and frequently fixed to the cell centre. However, they failed to divide or eject and were left at the new cell pole of one cell at division. Thus, ParB appears to be required for recognition of the plasmid and its attachment to the cell centre, and ParA is required for focus division and energetic ejection from the cell centre. The ATPase active site mutation, parAK122E, blocked ejection. Mutant parAM314I ejected weakly, and the daughter foci took two generations to reach a new cell centre. This explains the novel alternation of segregation and missegregation in successive generations seen in time-lapse images of this mutant.
类parS着丝粒位点在存在P1 ParA和ParB蛋白的情况下促进P1质粒的主动分离。在此处使用的适度生长速率下，延时和静态显微镜观察表明，质粒拷贝在大肠杆菌细胞中心聚集成一个焦点。就在细胞分裂前，该焦点被主动分开并双向喷射到分裂细胞的相对两半中。在没有野生型parS结合蛋白ParB的情况下，形成了一个焦点，但通常不会移至细胞中心。随机放置的焦点不会分裂，仅由一个子细胞继承。在没有ParA的情况下，形成了焦点并经常固定在细胞中心。然而，它们未能分裂或喷射，在分裂时留在一个细胞的新细胞极处。因此，ParB似乎是识别质粒及其附着到细胞中心所必需的，而ParA是焦点分裂和从细胞中心有力喷射所必需的。ATP酶活性位点突变parAK122E阻止了喷射。突变体parAM314I喷射较弱，子代焦点需要两代才能到达新的细胞中心。这解释了在该突变体的延时图像中连续几代中观察到的分离和错误分离的新交替现象。
