Shibata T, DasGupta C, Cunningham R P, Williams J G, Osber L, Radding C M
J Biol Chem. 1981 Jul 25;256(14):7565-72.
Purified recA protein, which is essential for genetic recombination of Escherichia coli, catalyzed ATP-dependent homologous pairing of double-stranded DNA and single-stranded fragments to form D-loops. When the double-stranded DNA was nicked circular DNA (form II) or linear DNA (form III), the reaction proceeded nearly linearly during 30 min of incubation at 37 degrees C. When the double-stranded DNA was superhelical (form I), anomalous kinetics was observed. This anomaly was suppressed by the addition of spermidine without affecting the final yield of D-loops. The formation of D-loops required stoichiometric amounts of recA protein, which were proportional to the concentration of single-stranded DNA but which were not affected by the concentration of double-stranded DNA. With form II or III DNA as the recipient for the formation of D-loops, the rate of the reaction was greatest when there was one monomer of recA protein/2-3 nucleotide residues of single-stranded DNA; larger amounts of single-stranded DNA inhibited the reaction. The formation of D-loops was half inhibited by 30 mM NaCl and by 0.6 mM ADP, one of the products of the reaction. The thermal stability of D-loops made by recA protein was the same as that of D-loops made by annealing. In addition to pairing linear single strands with duplex DNA, recA protein made joint molecules from single-stranded circular DNA and homologous form II or III DNA. According to these and previous observations (Cunningham, R. P., DasGupta, C., Shibata, T., and Radding, C. M. (1980) Cell 20, 223-235), rcA protein will stably pair two molecules of DNA if one of them is single-stranded or partially single-stranded and if either molecule has a free end.
纯化的RecA蛋白对大肠杆菌的基因重组至关重要，它催化ATP依赖的双链DNA与单链片段的同源配对以形成D环。当双链DNA为带切口的环状DNA（II型）或线性DNA（III型）时，在37℃孵育30分钟期间反应几乎呈线性进行。当双链DNA为超螺旋（I型）时，观察到异常动力学。添加亚精胺可抑制这种异常，且不影响D环的最终产量。D环的形成需要化学计量的RecA蛋白，其与单链DNA的浓度成正比，但不受双链DNA浓度的影响。以II型或III型DNA作为形成D环的受体时，当存在1个RecA蛋白单体/2 - 3个单链DNA核苷酸残基时反应速率最大；单链DNA量增加会抑制反应。30 mM NaCl和0.6 mM ADP（反应产物之一）可使D环的形成受到一半抑制。RecA蛋白形成的D环的热稳定性与退火形成的D环相同。除了使线性单链与双链DNA配对外，RecA蛋白还能使单链环状DNA与同源II型或III型DNA形成连接分子。根据这些及先前的观察结果（坎宁安，R.P.，达斯古普塔，C.，柴田，T.，和拉丁，C.M.（1980年）《细胞》20卷，223 - 235页），如果其中一个DNA分子是单链或部分单链且任一分子有自由末端，RecA蛋白会稳定地使两个DNA分子配对。
