Breier Adam M, Weier Heinz-Ulrich G, Cozzarelli Nicholas R
Graduate Group in Biophysics and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):3942-7. doi: 10.1073/pnas.0500812102. Epub 2005 Feb 28.
In Escherichia coli DNA replication is carried out by the coordinated action of the proteins within a replisome. After replication initiation, the two bidirectionally oriented replisomes from a single origin are colocalized into higher-order structures termed replication factories. The factory model postulated that the two replisomes are also functionally coupled. We tested this hypothesis by using DNA combing and whole-genome microarrays. Nascent DNA surrounding oriC in single, combed chromosomes showed instead that one replisome, usually the leftward one, was significantly ahead of the other 70% of the time. We next used microarrays to follow replication throughout the genome by measuring DNA copy number. We found in multiple E. coli strains that the replisomes are independent, with the leftward replisome ahead of the rightward one. The size of the bias was strain-specific, varying from 50 to 130 kb in the array results. When we artificially blocked one replisome, the other continued unabated, again demonstrating independence. We suggest an improved version of the factory model that retains the advantages of threading DNA through colocalized replisomes at about equal rates, but allows the cell flexibility to overcome obstacles encountered during elongation.
在大肠杆菌中，DNA复制是由复制体中的蛋白质协同作用来完成的。复制起始后，来自单一复制起点的两个双向复制体共定位形成称为复制工厂的高阶结构。工厂模型推测这两个复制体在功能上也是耦合的。我们通过使用DNA梳理和全基因组微阵列来检验这一假设。相反，在单个梳理后的染色体中，oriC周围的新生DNA显示，一个复制体，通常是向左的那个，在70%的时间里明显领先于另一个。接下来，我们使用微阵列通过测量DNA拷贝数来跟踪整个基因组的复制情况。我们在多个大肠杆菌菌株中发现，复制体是独立的，向左的复制体领先于向右的复制体。偏差的大小具有菌株特异性，在阵列结果中从50到130 kb不等。当我们人工阻断一个复制体时，另一个复制体仍继续不受影响地进行，再次证明了其独立性。我们提出了一个改进版的工厂模型，该模型保留了以大致相同的速率使DNA穿过共定位的复制体的优点，但允许细胞具有灵活性，以克服延伸过程中遇到的障碍。
