Santamaría D, de la Cueva G, Martínez-Robles M L, Krimer D B, Hernández P, Schvartzman J B
Departamento de Biología Celular y del Desarrollo, CIB (Consejo Superior de Investigaciones Científicas), Velázquez 144, 28006 Madrid, Spain.
J Biol Chem. 1998 Dec 11;273(50):33386-96. doi: 10.1074/jbc.273.50.33386.
A series of plasmids were constructed containing two unidirectional ColE1 replication origins in either the same or opposite orientations and their replication mode was investigated using two-dimensional agarose gel electrophoresis. The results obtained showed that, in these plasmids, initiation of DNA replication occurred at only one of the two potential origins per replication round regardless of origins orientation. In those plasmids with inversely oriented origins, the silent origin act as a polar pausing site for the replication fork initiated at the other origin. The distance between origins (up to 5.8 kilobase pairs) affected neither the interference between them to initiate replication nor the pausing function of the silent origin. A deletion analysis indicated that the presence of a transcription promoter upstream of the origin was the only essential requirement for it to initiate replication as well as to account for its polar pausing function. Finally, in vitro helicase assays showed that Escherichia coli DnaB is able to melt DNA-DNA homoduplexes but is very inefficient to unwind RNA-DNA hybrids. Altogether, these observations strongly suggest that replication forks pause at silent ColE1 origins due to the inability of DnaB helicase, which leads the replication fork in vivo, to unwind RNA-DNA hybrids.
构建了一系列质粒，这些质粒含有两个同向或反向的单向ColE1复制起点，并使用二维琼脂糖凝胶电泳研究了它们的复制模式。所得结果表明，在这些质粒中，每轮复制中DNA复制起始仅发生在两个潜在起点中的一个，而与起点方向无关。在那些起点反向的质粒中，沉默起点作为从另一个起点起始的复制叉的极性暂停位点。起点之间的距离（长达5.8千碱基对）既不影响它们之间起始复制的干扰，也不影响沉默起点的暂停功能。缺失分析表明，起点上游存在转录启动子是其起始复制以及解释其极性暂停功能的唯一必要条件。最后，体外解旋酶分析表明，大肠杆菌DnaB能够解开DNA-DNA同源双链体，但解开RNA-DNA杂种的效率非常低。总之，这些观察结果强烈表明，复制叉在沉默的ColE1起点处暂停是由于在体内引导复制叉的DnaB解旋酶无法解开RNA-DNA杂种。
