Craigie R, Arndt-Jovin D J, Mizuuchi K
Proc Natl Acad Sci U S A. 1985 Nov;82(22):7570-4. doi: 10.1073/pnas.82.22.7570.
An early step in the transposition of bacteriophage Mu DNA in vitro is a DNA strand-transfer reaction that generates an intermediate DNA structure in which the Mu donor DNA and the target DNA are covalently joined. DNA replication, initiated at the DNA forks in this intermediate, generates a cointegrate product; simple insert products can also be formed from the same intermediate by degradation of a specific segment of the structure, followed by gap repair. This DNA strand-transfer reaction requires ATP, magnesium, the Mu A and Mu B proteins, and a factor supplied by an Escherichia coli cell extract. We have now shown that the host protein factor requirement can be satisfied by purified protein HU. The defined system has been used to determine the DNA substrate requirements for the reaction. The reaction requires the two Mu ends, located on the same DNA molecule, in the same relative orientation to one another as in the phage Mu genome. To participate in the strand-transfer reaction efficiently the mini-Mu plasmid, used as the transposon donor, must be supercoiled; the target DNA molecule may be supercoiled, relaxed circular, or linear.
噬菌体Mu DNA体外转座的早期步骤是一个DNA链转移反应，该反应产生一种中间DNA结构，其中Mu供体DNA和靶DNA共价连接。在这个中间体的DNA叉处起始的DNA复制产生一个共整合产物；简单的插入产物也可以由相同的中间体通过结构特定片段的降解，随后进行缺口修复而形成。这个DNA链转移反应需要ATP、镁、Mu A和Mu B蛋白，以及大肠杆菌细胞提取物提供的一个因子。我们现在已经表明，宿主蛋白因子的需求可以由纯化的HU蛋白满足。这个明确的系统已被用于确定该反应对DNA底物的要求。该反应需要位于同一DNA分子上的两个Mu末端，它们彼此之间的相对方向与噬菌体Mu基因组中的相同。作为转座子供体的微型Mu质粒要有效地参与链转移反应，必须是超螺旋的；靶DNA分子可以是超螺旋的、松弛环状的或线性的。
