Champoux J J, McConaughy B L
Biochemistry. 1975 Jan 28;14(2):307-16. doi: 10.1021/bi00673a017.
When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA. The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA. The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides. The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template. The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E. coli DNA polymerase I. After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized. During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis. This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix. Possible models for in vivo priming of double-stranded DNA by E. coli RNA polymerase are discussed.
当含有58个负超螺旋圈的闭环SV40 DNA用作大肠杆菌RNA聚合酶进行RNA合成的模板时，一部分RNA产物会与DNA保持复合状态。复合物中的RNA在高盐条件下对核糖核酸酶具有抗性，其解链温度表明它与DNA形成了氢键。复合物中RNA与DNA核苷酸的摩尔比在0.01至0.08之间；RNA长度在80至600个核苷酸之间。复合物的形成依赖于环状DNA在拓扑学上的解旋，因为当使用含有零个超螺旋圈的闭环DNA作为模板时不会形成复合物。DNA-RNA复合物可作为大肠杆菌DNA聚合酶I进行体外DNA合成的引物-模板组合。在用（α-32P）标记的脱氧核糖核苷三磷酸进行合成并随后进行碱水解后，分离出32P标记的核糖核苷酸证明了RNA与合成的DNA之间存在共价连接。在体外DNA合成过程中，模板以较低速率产生切口，有切口的分子支持大量的DNA合成。这一观察结果表明，未切口的分子可能由于螺旋解旋的拓扑学限制而只能进行有限的合成。文中还讨论了大肠杆菌RNA聚合酶在体内对双链DNA进行引发的可能模型。
