Register J C, Griffith J
J Biol Chem. 1985 Oct 5;260(22):12308-12.
The RecA protein of Escherichia coli optimally promotes DNA strand exchange reactions in the presence of the single strand DNA-binding protein of E. coli (SSB protein). Under these conditions, assembly of RecA protein onto single-stranded DNA (ssDNA) occurs in three steps. First, the ssDNA is rapidly covered by SSB protein. The binding of RecA protein is then initiated by nucleation of a short tract of RecA protein onto the ssDNA. Finally, cooperative polymerization of additional RecA protein accompanied by displacement of SSB protein results in a ssDNA-RecA protein filament (Griffith, J. D., Harris, L. D., and Register, J. C. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559). We report here that RecA protein assembly onto circular ssDNA yields RecA protein-covered circles in which greater than 85% are completely covered by RecA protein with no remaining SSB protein-covered segments (as detected by electron microscopy). However, when linear ssDNA is used, 90% of the filaments contain a short segment at one end complexed with SSB protein. This suggests that RecA protein assembly is unidirectional. Visualization of the assembly of RecA protein onto either long ssDNA tails (containing either 5' or 3' termini) or ssDNA gaps generated in double strand DNA allowed us to determine that the RecA protein polymerizes in the 5' to 3' direction on ssDNA and preferentially nucleates at ssDNA-double strand DNA junctions containing 5' termini.
大肠杆菌的RecA蛋白在大肠杆菌单链DNA结合蛋白（SSB蛋白）存在的情况下，能最佳地促进DNA链交换反应。在这些条件下，RecA蛋白组装到单链DNA（ssDNA）上的过程分为三步。首先，ssDNA迅速被SSB蛋白覆盖。然后，一小段RecA蛋白在ssDNA上成核，从而启动RecA蛋白的结合。最后，伴随SSB蛋白被取代，额外RecA蛋白的协同聚合形成了ssDNA-RecA蛋白细丝（格里菲思，J.D.，哈里斯，L.D.，和雷吉斯特，J.C.（1984年）《定量生物学冷泉港研讨会》49，553 - 559）。我们在此报告，RecA蛋白组装到环状ssDNA上会产生被RecA蛋白覆盖的环，其中超过85%被RecA蛋白完全覆盖，没有剩余的被SSB蛋白覆盖的片段（通过电子显微镜检测）。然而，当使用线性ssDNA时，90%的细丝在一端含有与SSB蛋白复合的短片段。这表明RecA蛋白的组装是单向的。通过观察RecA蛋白在长ssDNA尾巴（含有5'或3'末端）或双链DNA中产生的ssDNA缺口上的组装过程，我们得以确定RecA蛋白在ssDNA上沿5'到3'方向聚合，并且优先在含有5'末端的ssDNA - 双链DNA连接处成核。
