Inciarte M R, Salas M, Sogo J M
J Virol. 1980 Apr;34(1):187-99. doi: 10.1128/JVI.34.1.187-199.1980.
We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.
我们从噬菌体感染的枯草芽孢杆菌提取物中分离出φ29 DNA复制中间体，用[3H]胸腺嘧啶脉冲标记，先在中性蔗糖中进行速度沉降，然后进行CsCl平衡密度梯度离心。在追踪过程中，在中性蔗糖中沉降系数较高而在碱性蔗糖中沉降速率低于病毒φ29 DNA的DNA转化为成熟DNA。通过电子显微镜分析，密度高于成熟φ29 DNA的物质由复制中间体组成。我们发现了两种主要类型的分子。一种由单位长度的双链DNA组成，在随机位置有一个单链分支。单链分支的长度与双链区域之一的长度相似。另一种类型的分子是单位长度的DNA，有一个双链区域和一个从一端延伸不同距离的单链区域。对后一种分子的部分变性表明，复制从DNA的任何一端开始的频率相似。这些发现表明，φ29 DNA复制通过链置换机制发生，并且复制从DNA的任何一端非同时开始，就像腺病毒的情况一样。
