Segregation of Bacterial Chromosomes to Daughter Cells

大肠杆菌有一个单一的圆形染色体，在细胞分裂过程中可以非常精确地复制和分离到子细胞。复制从单一起始点双向进行，并终止于染色体的另一侧。该系统的相对简单性和其繁殖所需的有限数量的细胞成分使其成为一般DNA复制和分离的模型系统。我们已经开发了一个P1 PARS GFP-PARB系统，用于通过荧光显微镜对大肠杆菌染色体上的任何所需位点进行定位。这项技术在活细胞中工作得很好，使我们能够通过延时显微镜跟踪几代人的染色体序列的命运。此外，我们还将该技术与流式细胞术相结合，用来确定细胞群体中细胞周期中特定位置的空间分布。我们目前有关于在简单细胞周期中以中等生长速度生长的细胞中分布在染色体周围的多倍体的动态行为的数据。我们的主要结论如下：1.染色体分离基本完成，复制仍在进行。末端在细胞中心复制，所有细胞的子代末端一直附着在那里，直到细胞分裂，当细胞分裂时，它们迅速彼此分离。当细胞接近分裂时，末端焦点位于真正的细胞中心：焦点与FtsZ环的一段共同定位。3.细胞中心末端的捕获不依赖于XerCD位点特异性重组系统。它似乎也独立于FtsK的C-末端结构域，FtsK是一种与细胞中心的DNA缠绕有关的蛋白质，尽管它在许多ftsK突变细胞中由于细胞分裂事件的异常或缺失而中断。4.一个160kb的末端结构域，以dif重组位点为中心，在细胞分裂时分离为一个单位。这一区域两侧的位置在细胞分裂之前是分离的。5.复制的起源在细胞周期中相当早地分离。平均而言，在起源起始和分离之间有大约四分之一的世代延迟。然而，不同细胞的延迟差别很大，一些起源在启动后立即分离。6.原点从细胞中心向两极分离，在附着到新的细胞中心之前可以自由移动一段时间。女儿的起源有时会在最初的分离后重新关联，然后再次分离。起源的凝聚力虽然经常发生，但不是细胞周期的一个必要或不变的特征。7.染色体周围位于终止域和起始区之间的位置在细胞分裂前分离，有时大致对应于它们的图谱位置。有一些位置的平均分离时间比由复制顺序计时的标记的有序分离预测的更早。这些地区可能是由隔离机制以某种特殊方式处理的地区。今年，我们特别强调了染色体分离机制的一个组成部分--SEQA蛋白。SEQA是一种当腺嘌呤碱基半甲基化时与GATC序列特异结合的蛋白质。这种底物是在染色体基因座新复制时产生的。SEQA短暂地与新复制的DNA结合，该DNA跟随复制分叉的进程。我们有证据表明，SEQA结合以一种对适当的DNA分离很重要的方式组织和压缩新复制的DNA。书名/作者The Heavy of the China in the China)/作者J.我们已经解决了SEQA蛋白的大部分结构，并利用结构信息来探索其功能。我们发现SEQA形成了一条螺旋状的丝状结构，新复制的DNA被包裹在上面。通过将二聚体添加到新复制的GATC序列中，细丝从复制叉处生长。在细丝的另一端，当DNA完全甲基化时，二聚体就会丢失。因此，DNA的一束在与SEQA细丝结合的染色体周围进行。这种DNA组织似乎在复制过程中染色体的分离中发挥了核心作用。细菌中染色体分离的总体机制显然与高等生物中有丝分裂的机制截然不同。然而，它与将真核生物中新复制的DNA分离成不同的姐妹染色单体的机制可能有很大的相似之处。

The bacterium Escherichia coli has a single, circular chromosome that is replicated and segregated with great precision to daughter cells during cell division. Replication proceeds bi-directionally from a single origin and terminates on the opposite side of the chromosome. The reletive simplicity of this system and the limited number of cell components required for its propagation make it a model system for DNA replication and segregation in general. We have developed a P1 parS GFP-ParB system for localization by fluorescent microscopy of any desired locus on the E. coli chromosome. The technique works well in living cells and allows us to follow the fate of chromosomal sequences through several generations by time-lapse microscopy. In addition, we have used the technique, in combination with flow cytometry, to determine the spatial distributions of given loci at defined points in the cell cycle in a cell population. We currently have data for the dynamic behavior of multipleloci distributed around the chromosome in cells growing with a simple cell cycle at moderate growth rates. Our principle conclusions are as follows:1. Chromosome segregation is primarily accomplished while replication is still ongoing.2. The terminus is replicated at the cell center and the daughter termini of all cells remain attached there until cell division, at which time they rapidly segregate away from each other as the cell divides. The terminal foci are at the true cell center as the cell approaches division : the foci co-localize with a segment of the FtsZ ring. 3. The capture of the termini at the cell center is independent of the xerCD site-specific recombination system. It also appears to be independent of the C-terminal domain of FtsK, a protein implicated in DNA hadling at the cell center, although it is disrupted in many of the ftsK mutant cells due to aberrant or absent cell division events. 4. A terminus domain of 160kb., centered on the dif recombination site, segregates as a unit at cell division. Positions flanking this region segregate prior to cell division. 5. Origins of replication segregate fairly early in the cell cycle. On average, there is a delay of about 1/4 of a generation between origin initiation and segregation. However, the delay varies widely from cell to cell, with some origins segregating immediately after initiation. 6. Origins segregate from the cell center toward the poles and are free to move about for some time before becoming attached to the new cell centers. Daughter origins sometimes re-associate after initial segregation and dissociate again. Cohesion of origins, although it often occurs, is not a necessary or invariant feature of the cell cycle. 7. Positions around the chromosome that are intermediate between the terminal domain and the origin segregate before cell division, at times roughly corresponding to their map positions. There are some positions whose average segregation time appears earlier that predicted by an orderly segregation of markers timed by replication order. These may be regions that are handled in some special way by the segregation machinery. This year, we have put special emphasis on one component of the chromosome segregation machinery, the SeqA protein. SeqA is a protein that binds specifically to the sequence GATC when the adenine bases are hemi-methylated. This substrate is produced when chromosome loci are newly replicated. SeqA binds transiently to the newly replicated DNA that follows the progression of the replication forks. We have evidence that SeqA binding organizes and compacts the newly replicated DNA in a way that is important for proper DNA segregation. In collaboration with Wei Yang (NIDDK), and Alba Guarne (McMaster U.) we have solved the structure of much of the SeqA protein, and used the structural information to probe its function. We find that SeqA forms a helical filament on which the newly replicated DNA is wrapped. The filament grows from the replication fork by addition of dimers to the newly replicated GATC sequences. At the other end of the filament, dimers are lost as the DNA becomes fully methylated. Thus, a tract of the DNA progresses around the chromosome bound to a SeqA filament. This DNA organization appears to play a central role in segregation of the chromosome during replication. The overall mechanism for chromosome segregation in bacteria is clearly quite distinct from that of mitosis in higher organisms. However, there may be a strong resemblance between it and the mechanism that segregates the newly replicated DNA in eucaryotes into distinct sister chromatids.