Imae Y, Okazaki R
J Virol. 1976 Aug;19(2):435-45. doi: 10.1128/JVI.19.2.435-445.1976.
A new in vitro system for T4 DNA replication was developed by concentrating cell lysates on cellophane disks. The time course of [3H]dTTP incorporation into DNA by the system was separated into two phases: one was a very rapid incorporation which was terminated within 2 min (phase I reaction), and the other was a slow but continuous incorporation thereafter (phase II reaction). More than half of the phase I reaction product was Escherichia coli DNA, but the phase II reaction was mostly T4 DNA. Phase II reaction required four deoxyribonucleoside triphosphates, ATP, Mg2+, and KCl. 5-Hydroxymethyldeoxycytidine triphosphate was essential for the reaction and not substitutable by dCTP. The presence of KCN or NaN3 in the reaction mixture did not interfere with [3H]dTTP incorporation, but the addition of deoxyribonuclease completely degraded the system. Alkaline sucrose sedimentation analysis of phage II reaction product revealed that phase II reaction proceeded by the discontinuous mode of DNA replication as in vivo. After T4 infection, the activity for phase II reaction appeared in parallel with the activity of T4 phage DNA replication in vivo.
通过将细胞裂解物浓缩在玻璃纸圆盘上，开发了一种用于T4 DNA复制的新体外系统。该系统将[3H]dTTP掺入DNA的时间进程分为两个阶段：一个是非常快速的掺入，在2分钟内终止（I期反应），另一个是此后缓慢但持续的掺入（II期反应）。I期反应产物的一半以上是大肠杆菌DNA，但II期反应主要是T4 DNA。II期反应需要四种脱氧核糖核苷三磷酸、ATP、Mg2+和KCl。5-羟甲基脱氧胞苷三磷酸对该反应至关重要，不能被dCTP替代。反应混合物中存在KCN或NaN3不会干扰[3H]dTTP的掺入，但加入脱氧核糖核酸酶会完全破坏该系统。对噬菌体II期反应产物的碱性蔗糖沉降分析表明，II期反应如在体内一样以DNA复制的不连续模式进行。T4感染后，II期反应的活性与体内T4噬菌体DNA复制的活性平行出现。
