Takahashi M, Nordén B
Groupe d'Etude Mutagénèse et Cancérogénèse, URA 1342 CNRS, Institut Curie, Université Paris Sud, Orsay, France.
Adv Biophys. 1994;30:1-35. doi: 10.1016/0065-227x(94)90009-4.
The importance of filament formation of RecA for the DNA strand exchange reaction, both in vivo and in vitro, is established. RecA forms a very long and relatively stiff filament by binding around DNA with high cooperativity. The monomer units are assembled in the filament in a head-to-tail arrangement in a helical manner, similar to the organization of RecA molecules found in the crystal of pure RecA or including ADP. This filament of RecA, containing a DNA molecule in its interior, can bind another DNA molecule and yet a third one in the presence of cofactor (ATP or its analogs). Each filament may have three DNA binding sites, each able to bind one DNA strand of either ss or ds DNA. According to linear dichroism and fluorescence spectroscopies, the DNA molecules in the RecA filament are well organized with a well defined but modified structure. This organization and modification of DNA by RecA probably has the purpose of facilitating Watson-Crick base-pair recognition and strand exchange reaction. RecA is thus actively involved in the reaction. The phosphoribose backbone of DNA follows the RecA helix and the DNA is stretched 50% and unwound. The nucleobases are destacked but still firmly oriented almost perpendicular to the axis for the first DNA and are immobile even in the case of ssDNA. Even in the second DNA the motion of DNA bases is very restricted although their orientation appears to be less perpendicular. All DNA strands in the complex show some sequence dependent interaction with each other. It could be a non-conventional base-base pairing, that could provide a mechanism of search for homologous DNA. In order to understand how RecA organizes DNA, a 3-D model building of the RecA-DNA complex is in progress undertaken based on the crystal structure of RecA and results obtained using chemical interference and protein engineering techniques. A characterization of structure of the second DNA and the mode of DNA-DNA interaction may further clarify the reaction mechanism of strand exchange.
RecA形成丝状结构对于体内和体外DNA链交换反应的重要性已得到证实。RecA通过与DNA高度协同结合形成非常长且相对刚性的丝状结构。单体单元以螺旋方式首尾相连排列在丝状结构中，类似于在纯RecA晶体或包含ADP的RecA晶体中发现的RecA分子的组织方式。这种内部含有一个DNA分子的RecA丝状结构，在辅因子（ATP或其类似物）存在的情况下，可以结合另一个DNA分子，甚至第三个DNA分子。每个丝状结构可能有三个DNA结合位点，每个位点都能够结合单链或双链DNA的一条DNA链。根据线性二色性和荧光光谱学，RecA丝状结构中的DNA分子组织良好，具有明确但经过修饰的结构。RecA对DNA的这种组织和修饰可能旨在促进沃森-克里克碱基对识别和链交换反应。因此，RecA积极参与该反应。DNA的磷酸核糖主链沿着RecA螺旋延伸，DNA被拉伸50%并解旋。核碱基堆积被破坏，但仍然几乎垂直于第一条DNA的轴牢固地定向，即使在单链DNA的情况下也是固定不动的。即使在第二条DNA中，DNA碱基的运动也受到很大限制，尽管它们的定向似乎不太垂直。复合物中的所有DNA链彼此之间都表现出一些序列依赖性相互作用。这可能是一种非常规的碱基对配对，可能提供一种寻找同源DNA的机制。为了了解RecA如何组织DNA，正在基于RecA的晶体结构以及使用化学干扰和蛋白质工程技术获得的结果进行RecA-DNA复合物的三维模型构建。对第二条DNA的结构表征以及DNA-DNA相互作用模式可能会进一步阐明链交换的反应机制。
