Müller B, Tsaneva I R, West S C
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts, United Kingdom.
J Biol Chem. 1993 Aug 15;268(23):17185-9.
Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro. The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB. The presence of E. coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179-17184). To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I. The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction. RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA. In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner. At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.
利用RecA蛋白产生的重组中间体，我们已经证明大肠杆菌RuvB蛋白能够在体外介导霍利迪连接体的分支迁移。该反应依赖于存在≥10 mM的Mg2+以及化学计量的RuvB。大肠杆菌RuvA蛋白的存在降低了对Mg2+的需求以及对RuvB的化学计量需求（Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179 - 17184）。为了确定这两种蛋白在分支迁移过程中的作用，我们通过以下方法研究了在不存在或存在RuvA的情况下RuvB与DNA的相互作用：(i) 蛋白质 - DNA复合物的凝胶阻滞分析，(ii) RuvB ATP酶活性的刺激，以及(iii) DNA对DNase I的抗性。RuvB与双链DNA的相互作用依赖于Mg(2+)，并且与RuvB介导的分支迁移反应对Mg2+的需求相关。RuvB也与单链DNA相互作用，但其亲和力明显低于双链DNA。与RuvB不同，RuvA与双链DNA的相互作用在不存在Mg2+的情况下发生，并且以浓度依赖的方式受到Mg2+的抑制。在5 mM Mg2+时，RuvA蛋白促进了RuvB与DNA的相互作用，导致形成包含RuvA、RuvB和双链DNA的复合物。
