Martín-Parras L, Hernández P, Martínez-Robles M L, Schvartzman J B
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
J Biol Chem. 1992 Nov 5;267(31):22496-505.
We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.
我们研究了含有多个潜在复制起点的细菌质粒中复制起点的使用频率。用pBR322单体、二聚体或三聚体对大肠杆菌recA-细胞进行选择性转化。分离质粒DNA并用一种仅切割单体一次的限制性酶进行消化，然后通过中性/中性二维琼脂糖凝胶电泳分析复制中间体（RI）。仅在寡聚体中发现了线性化质粒外部起始的证据。此外，在二聚体中，指示外部起始的信号强度与反映内部起始的信号强度相当，而在三聚体中，前者约为后者的两倍。为了确定在单个质粒中两个起点是否能同时起始复制，我们研究了一种新二聚体的复制，其中两个单元可以明确区分。结果表明，虽然两个起点都同样有能力起始复制，但每个质粒只有一个是活跃的。这些观察结果强烈表明，在ColE1质粒中，即使每个质粒有几个相同的潜在起点，复制也在单个位点起始。除了传统的二维凝胶模式外，还观察到了新颖的特定模式，其强度因DNA样品而异。这些独特的模式是RI在复制叉处断裂的结果。这种类型的断裂会改变RI的质量和形状。当整个RI群体受到影响时，会形成一个新的分子群体，可能在二维凝胶中产生新的模式。
