Kim S K, Nordén B, Takahashi M
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
J Biol Chem. 1993 Jul 15;268(20):14799-804.
RecA protein can bind to double-stranded DNA even without the cofactor ATP if a DNA intercalator such as ethidium bromide is present (Thresher R. J., and Griffith, J. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5056-5060). We have studied the structure and association kinetics of the ethidium-promoted DNA-RecA complex in order to understand the role of this intercalator in the DNA-RecA association process, information that could provide insight about the binding mechanism of RecA to DNA. Both linear dichroism and fluorescence measurements show that ethidium remains intercalated between the DNA bases in the RecA-DNA complex in the absence of ATP. Even in the presence of the ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ethidium bromide shows some stimulating effect on the binding of RecA to DNA. The results indicate that the destacking of DNA bases is an important limiting step in the association of RecA to DNA (DNA is stretched in the ATP gamma S-RecA-DNA complex). In the presence of ATP gamma S, however, ethidium was extruded from DNA upon the binding of RecA. This result suggests that the binding mechanism of RecA to DNA may involve intercalation of one or more amino acid residues of RecA between the DNA bases. Such an intercalation would also be consistent with the stretching of DNA and the observation that the DNA bases remain in a (virtually stacked) perpendicular geometry (Takahashi, M., Kubista, M., and Nordén, B. (1991) Biochemie (Paris) 73, 219-226; Nordén, B., Elvingson, C., Kubista, M., Sjöberg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M. (1992b) J. Mol. Biol. 226, 1175-1191).
如果存在诸如溴化乙锭之类的DNA嵌入剂，即使没有辅助因子ATP，RecA蛋白也能与双链DNA结合（Thresher R. J.和Griffith, J. D.（1990年）《美国国家科学院院刊》87, 5056 - 5060）。我们研究了溴化乙锭促进的DNA - RecA复合物的结构和缔合动力学，以便了解这种嵌入剂在DNA - RecA缔合过程中的作用，这些信息可能有助于深入了解RecA与DNA的结合机制。线性二色性和荧光测量均表明，在没有ATP的情况下，溴化乙锭仍嵌入RecA - DNA复合物的DNA碱基之间。即使存在ATP类似物腺苷5'-O-(3 - 硫代三磷酸)（ATPγS），溴化乙锭对RecA与DNA的结合也有一定的刺激作用。结果表明，DNA碱基的解堆叠是RecA与DNA缔合过程中的一个重要限制步骤（在ATPγS - RecA - DNA复合物中DNA被拉伸）。然而，在存在ATPγS的情况下，RecA结合后溴化乙锭从DNA中被挤出。这一结果表明，RecA与DNA的结合机制可能涉及RecA的一个或多个氨基酸残基嵌入DNA碱基之间。这种嵌入也与DNA的拉伸以及DNA碱基保持（几乎堆叠的）垂直几何形状的观察结果一致（Takahashi, M., Kubista, M., and Nordén, B.（1991年）《生物化学（巴黎）》73, 219 - 226；Nordén, B., Elvingson, C., Kubista, M., Sjöberg, B., Ryberg, H., Ryberg, M., Mortensen, K., and Takahashi, M.（1992b年）《分子生物学杂志》226, 1175 - 1191）。
