Taljanidisz J, Decker R S, Guo Z S, DePamphilis M L, Sarkar N
Department of Metabolic Regulation, Boston Biomedical Research Institute, MA 02114.
Nucleic Acids Res. 1987 Oct 12;15(19):7877-88. doi: 10.1093/nar/15.19.7877.
Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA.
被猿猴病毒40（SV40）感染的CV - 1细胞的无细胞提取物，能够在含有功能性SV40复制起点（ori）的环状DNA分子中启动大肿瘤抗原依赖性的双向复制。为了确定在这些条件下的DNA复制是否涉及RNA引发的DNA合成，在5 - 巯基脱氧胞苷三磷酸存在的情况下进行复制，以标记新生DNA链。通过其对硫醇 - 琼脂糖的亲和力分离新合成的汞化DNA，并对分离链的5'端进行放射性标记，以便鉴定RNA引物。至少50%的分离链在其5'端含有4至7个共价连接的核糖核苷酸；80%的寡核糖核苷酸以腺苷开头，19%以鸟苷开头。约60%的新生DNA链与SV40 ori区域退火，并且这些链中约80%是与早期mRNA相同的方向合成的。这些结果与SV40 DNA在体内复制的特性一致，并支持一种SV40 DNA复制起始模型，其中DNA引发酶在ori编码早期mRNA的那条链上启动DNA合成。
