Romano L J, Tamanoi F, Richardson C C
Proc Natl Acad Sci U S A. 1981 Jul;78(7):4107-11. doi: 10.1073/pnas.78.7.4107.
The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.
噬菌体T7 DNA复制的主要起始位点位于T7 DNA分子左端15%的距离处。这个基因间隔区富含A+T，包含一个单一的基因4蛋白识别位点，并且在两个T7 RNA聚合酶[RNA核苷酸转移酶（DNA定向），EC 2.7.7.6]的串联启动子之前。电子显微镜分析表明，T7 DNA聚合酶[DNA核苷酸转移酶（DNA定向），EC 2.7.7.7]和基因4蛋白在双链DNA上随机定位的切口处起始DNA合成，以产生分支分子。然而，加入T7 RNA聚合酶和核糖核苷三磷酸后，14%的产物分子有复制泡，所有这些复制泡都位于体内观察到的主要起始位点附近；在缺乏主要起始位点的T7缺失突变体LG37 DNA上没有发生这样的起始。我们还利用插入了T7 DNA片段的质粒研究了起始过程。这些模板上的DNA合成也依赖于T7 RNA聚合酶和核糖核苷三磷酸的存在。只要首先转化为线性形式，DNA合成对于含有主要起始位点的质粒是特异性的。
