Padmanabhan R
Biochemistry. 1977 May 3;16(9):1996-2003. doi: 10.1021/bi00628a037.
A pyrimidine octanucleotide complementary to one of the cohesive ends of P2 DNA was chemically synthesized. Its sequence, d(C-T-T-T-C-C-C-C-OH), was verified by labeling it at the 5' end, followed by partial enzyme digestion and separation by a two-dimensional fingerprinting system. A single ribo-G residue was added to its 3' end using calf thymus deoxynucleotidyl terminal transferase. The resulting nonanucleotide primer was used in a detailed study on the stability of the duplexes formed in the partial as well as complete repair synthesis catalyzed by DNA polymerase I, at 5 degrees C in the presence of 70 mM potassium phosphate and 70 mM NaCl. The nonanucleotide primer was able to form a stable duplex with P2 DNA template only in the presence of DNA polymerase I. When the chain lengths of pyrimidine oligonucleotides were varied from 4 to 8 to test their abilities to serve as primers for the enzymatic repair synthesis, it was revealed that the minimum length required for the primer function is 8. Using the nonanucleotide as the primer and the right-hand cohesive end of the DNA as the template, repair synthesis was initiated simultaneously at the 3' end of the primer as well as at the right-hand 3' end of the DNA. This resulted in a decrease in the efficiency of repair synthesis at the 3' end of the primer, possibly due to the displacement of the primer by the enzyme. The enzyme was unable to displace the primer, when the primer was extended to a 13-mer prior to the initiation of repair synthesis at the 3'-OH end of the DNA. These data suggest that the strand displacement by DNA polymerase I at 5 degrees C in the presence of 70 mM potassium phosphate and 70 mM NaCl is not significant when the duplex is at least 13 nucleotides long. The efficiency of the repair synthesis at the 3'-OH end of the DNA-primer duplex could be increased by blocking the repair synthesis at the 3'-OH end of the DNA by converting it to 3'- phosphate. This method could be useful in DNA sequence analysis, where such specific repair synthesis is desired.
化学合成了一种与P2 DNA粘性末端之一互补的嘧啶八核苷酸。通过在其5′末端进行标记，随后进行部分酶切并通过二维指纹图谱系统分离，验证了其序列d（C-T-T-T-C-C-C-C-OH）。使用小牛胸腺脱氧核苷酸末端转移酶在其3′末端添加了一个核糖-G残基。所得的九核苷酸引物用于详细研究在5℃、存在70 mM磷酸钾和70 mM氯化钠的条件下，DNA聚合酶I催化的部分及完全修复合成中形成的双链体的稳定性。仅在存在DNA聚合酶I的情况下，九核苷酸引物才能与P2 DNA模板形成稳定的双链体。当嘧啶寡核苷酸的链长从4变化到8以测试它们作为酶促修复合成引物的能力时，发现引物功能所需的最短长度为8。以九核苷酸为引物，以DNA的右手粘性末端为模板，在引物的3′末端以及DNA的右手3′末端同时启动修复合成。这导致引物3′末端的修复合成效率降低，可能是由于酶取代了引物。当在DNA的3′-OH末端启动修复合成之前将引物延伸至1３聚体时，该酶无法取代引物。这些数据表明，在5℃、存在70 mM磷酸钾和70 mM氯化钠的条件下，当双链体至少13个核苷酸长时，DNA聚合酶I的链置换作用不明显。通过将DNA的3′-OH末端转化为3′-磷酸来阻断其3′-OH末端的修复合成，可以提高DNA-引物双链体3′-OH末端的修复合成效率。该方法在需要进行此类特异性修复合成的DNA序列分析中可能有用。
