Selzer G, Tomizawa J I
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7082-6. doi: 10.1073/pnas.79.23.7082.
We report studies on the mechanism of initiation of DNA replication by p15A, a small plasmid whose origin of replication is known to function much as does that of ColE1. Previous work has shown that an RNA primer for DNA synthesis is generated by the action of RNase H (EC 3.1.26.4) on a precursor transcript. The precursor initiates well upstream of the origin of replication and somehow forms a hybrid with its template during transcription. Here we show that when RNase H cleaves the hybrid at 0 degrees C, an additional cleavage product besides the primer can be identified. Using two-dimensional RNA sequencing techniques, we have established the sequence of this product to within a few nucleotides of each end. The position of the 5' end indicates that the nuclease introduces a nick or very small gap in the precursor at the origin. This suggests that some sequence or structure directs the enzyme to the origin. The position of its 3' end indicates that the precursor terminates at or near a series of six dAs in the template strand about 190 nucleotides from the origin of replication. The data indicate that hybrid formation may be necessary for termination of the precursor at this downstream site.
我们报告了关于p15A引发DNA复制机制的研究，p15A是一种小质粒，其复制起点的功能与ColE1的复制起点非常相似。先前的研究表明，DNA合成的RNA引物是由核糖核酸酶H（EC 3.1.26.4）作用于前体转录本产生的。前体在复制起点上游很远的位置起始，并且在转录过程中以某种方式与其模板形成杂交体。在这里我们表明，当核糖核酸酶H在0摄氏度切割杂交体时，除了引物之外还能鉴定出一种额外的切割产物。使用二维RNA测序技术，我们已经确定了该产物两端各几个核苷酸范围内的序列。5'端的位置表明核酸酶在起点处的前体中引入了一个切口或非常小的缺口。这表明某些序列或结构将该酶导向起点。其3'端的位置表明前体在模板链中距复制起点约190个核苷酸处的一系列六个dA处或其附近终止。数据表明，杂交体的形成对于前体在这个下游位点的终止可能是必要的。
