Guiney D G, Helinski D R
J Biol Chem. 1975 Nov 25;250(22):8796-803.
The location of the protein in the open circular DNA form of the ColE1 DNA-protein relaxation complex, induced by treatment with sodium dodecyl sulfate, has been studied using several enzymes of DNA metabolism. Escherichia coli exonucleases I and III are able to degrade extensively the nicked strand of the relaxed complex from the 3' end. DNA polymerase I can initiate synthesis using the relaxed complex as template-primer and specifically extends the 3' end of the nicked strand. The 5' end of the sodium dodecyl sulfate-relaxed complex, however, is blocked to the 5'-3' hydrolitic action T7 exonuclease. This block remains after trypsin treatment of the sodium dodecyl sulfate-relaxed complex but is removed by Pronase treatment. T4 DNA ligase is unable to seal either the sodium dodecyl sulfate-relaxed complex or the Pronase-treated relaxed complex even after pretreatment of the relaxed complex with T4 DNA polymerase and polynucleotide kinase. However, pretreatment with DNA polymerase I and the four deoxyribonucleoside triphosphates facilitates ligase closure of the Pronase-treated relaxed complex but not the sodium dodecyl sulfate-relaxed complex. These studies indicate that the protein in the relaxed ColE1 complex is located at or near the 5' end of the nicked strand.
利用几种DNA代谢酶，研究了用十二烷基硫酸钠处理诱导产生的ColE1 DNA - 蛋白质松弛复合物的开放环状DNA形式中蛋白质的位置。大肠杆菌核酸外切酶I和III能够从3'端广泛降解松弛复合物的切口链。DNA聚合酶I可以以松弛复合物为模板 - 引物起始合成，并特异性地延伸切口链的3'端。然而，十二烷基硫酸钠 - 松弛复合物的5'端对T7核酸外切酶的5'-3'水解作用具有抗性。用胰蛋白酶处理十二烷基硫酸钠 - 松弛复合物后，这种抗性仍然存在，但经链霉蛋白酶处理后可被消除。即使在用T4 DNA聚合酶和多核苷酸激酶对松弛复合物进行预处理后，T4 DNA连接酶也无法封闭十二烷基硫酸钠 - 松弛复合物或链霉蛋白酶处理后的松弛复合物。然而，用DNA聚合酶I和四种脱氧核糖核苷三磷酸进行预处理有助于链霉蛋白酶处理后的松弛复合物的连接酶封闭，但对十二烷基硫酸钠 - 松弛复合物无效。这些研究表明，松弛的ColE1复合物中的蛋白质位于切口链的5'端或其附近。
