McMacken R, Kornberg A
J Biol Chem. 1978 May 10;253(9):3313-9.
Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome. Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription. The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together. Both RNA and DNA primers were initiated with ATP. At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers. Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme. At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates. Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues. The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template. The multienzyme priming system is highly specific for phiX174 DNA as template.
引发φX174单链DNA复制的寡核苷酸的合成利用了宿主细胞的复杂蛋白质机制，该机制可能被细胞用于复制其自身的染色体。引物合成至少依赖于五种蛋白质（DNA结合蛋白、dnaB和dnaC蛋白、蛋白i和蛋白n）以及ATP来形成复制中间体，还依赖于另一种蛋白质，即引发酶（dnaG蛋白），通过模板转录来组装寡核苷酸。本文中的数据表明，核糖核苷三磷酸和脱氧核糖核苷三磷酸都可以作为底物，当同时存在时会形成杂交引物。RNA和DNA引物均由ATP起始。转录生成有效引物需要四种碱基配对的核苷三磷酸中的至少三种。超过90%的RNA和DNA转录物被DNA聚合酶III全酶延伸成互补链。在最佳三磷酸浓度下，核糖核苷三磷酸形成引物的速率和程度比脱氧核糖核苷三磷酸更大。与DNA复制解偶联时，RNA引物的长度为14至50个残基，DNA引物为4至20个残基。RNA引物的RNase消化指纹图谱的复杂性表明是从φX174模板上的多个位点进行转录的。多酶引发系统对作为模板的φX174 DNA具有高度特异性。
