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):17179-84.
The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro. In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA. However, RuvA is required when the RuvB concentration is reduced 4-fold or more. Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides. To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB. The time courses of the two reactions were similar, and both required ATP and Mg2+. However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2). The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Müller, B., Tsaneva, I.R., and West, S. C. (1993) J. Biol. Chem. 268, 17185-17189) and can be overcome by addition of RuvA. These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.
大肠杆菌的RuvA和RuvB蛋白在体外介导Holliday连接体的分支迁移。在存在化学计量的RuvB（1个RuvB二聚体/12个核苷酸）时，分支迁移无需RuvA即可发生。然而，当RuvB浓度降低4倍或更多时，则需要RuvA。在最佳条件下，我们发现最小蛋白质需求量为每500 - 1100个核苷酸1个RuvB二聚体和每600 - 1200个核苷酸1个RuvA四聚体。为了确定RuvA和RuvB在分支迁移中的作用，我们比较了仅由RuvB介导以及由RuvA和RuvB介导的分支迁移反应。两种反应的时间进程相似，且都需要ATP和Mg²⁺。然而，RuvB介导的分支迁移在比RuvA和RuvB介导的反应（≥1 mM ATP，≥5 mM MgCl₂）更低的ATP浓度（≥200 μM）和更高的Mg²⁺浓度（≥10 mM MgCl₂）下发生。RuvB介导的分支迁移对Mg²⁺的需求反映了RuvB与DNA结合对Mg²⁺的需求（Müller，B.，Tsaneva，I.R.，和West，S.C.（1993）J. Biol. Chem. 268，17185 - 17189），并且可以通过添加RuvA来克服。这些结果表明RuvA蛋白促进了RuvB与DNA的相互作用。
