Ohmori H, Murakami Y, Nagata T
Institute for Virus Research, Kyoto University, Japan.
J Mol Biol. 1987 Nov 20;198(2):223-34. doi: 10.1016/0022-2836(87)90308-1.
To elucidate the replication mechanism of a ColE1-type plasmid in RNase H-deficient (rnh-) strains of Escherichia coli, we constructed plasmid derivatives that deleted the whole, or a part, of the 5'-AAAAA-3' sequence (positions -3 to +2) that acts as the origin of replication in vivo and in vitro in the presence of RNase H. The activity of plasmid replication in rnh+ cells was found to be reduced by alterations of the AAAAA sequence. The activity could be restored when the derivatives, retaining the upstream sequence down to -8, regained a sequence containing at least two A residues in the region from -3 to +2. By contrast, replication in rnh- cells was maintained at high levels even when the deletion included the AAAAA sequence and extended up to position -7. The activity in rnh- cells decreased as deletions proceeded to -8 and further up to -17, and was abolished completely by further upward deletions. We concluded that in rnh- cells the plasmid replicates by a mechanism that operates only when RNase H is inactive. This RNase H-sensitive replication in rnh- cells seems to require the RNA-DNA hybrid formation that is also required for RNase H-dependent replication in rnh+ cells. The hybrid formation probably contributes by unwinding a portion of DNA from which replication can be initiated.
为阐明ColE1型质粒在大肠杆菌核糖核酸酶H缺陷（rnh-）菌株中的复制机制，我们构建了质粒衍生物，这些衍生物缺失了5'-AAAAA-3'序列（位置-3至+2）的全部或部分，该序列在核糖核酸酶H存在的情况下在体内和体外作为复制起点。发现在rnh+细胞中，质粒复制活性因AAAAA序列的改变而降低。当保留至-8的上游序列的衍生物在-3至+2区域重新获得至少包含两个A残基的序列时，活性可以恢复。相比之下，即使缺失包括AAAAA序列并延伸至-7位置，rnh-细胞中的复制仍保持在高水平。随着缺失延伸至-8并进一步至-17，rnh-细胞中的活性降低，进一步向上缺失则完全消除了活性。我们得出结论，在rnh-细胞中，质粒通过一种仅在核糖核酸酶H无活性时起作用的机制进行复制。rnh-细胞中这种对核糖核酸酶H敏感的复制似乎需要RNA-DNA杂交体的形成，而rnh+细胞中依赖核糖核酸酶H的复制也需要这种杂交体的形成。杂交体的形成可能通过解开一部分可用于起始复制的DNA来发挥作用。
