Wittung P, Nordén B, Takahashi M
Department of Physical Chemistry, Chalmers University of Technology, Gothenburg, Sweden.
Eur J Biochem. 1994 Aug 15;224(1):39-45. doi: 10.1111/j.1432-1033.1994.tb19992.x.
To obtain mechanistic insights about RecA-promoted base pairing between complementary polynucleotides, the complex formation of RecA with poly(dA) and poly(dT) in the presence of ATP (and ATP-regenerating system) has been studied. The reaction was followed using a fluorescent probe, benzopyrenediolepoxide (BPDE), covalently attached to less than 1% of the adenine bases of poly(dA). BPDE is sensitive to its environment and has been found useful for detection of interactions between DNA strands, in the three binding positions of the RecA filament, in the presence of adenosine 5'-O-3-thiotriphosphate (ATP[S]) [Wittung, P., Nordén, B. & Takahashi, M. (1994) J. Biol. Chem. 269, 5799-5803]. The emission spectrum of RecA:BPDE-poly(dA) formed in the presence of ATP is similar to that observed with ATP[S] supporting similar structures of the complexes. However, the fluorescence anisotropy is considerably reduced, suggesting a higher degree of freedom of DNA in the presence of ATP hydrolysis. Upon addition of a complementary strand, poly(dT), to a preformed filament of RecA:BPDE-poly(dA) in the presence of ATP, the fluorescence intensity slowly decreases and a change of emission profile consistent with Watson-Crick base pairing is observed. This contrasts with the case of ATP[S] in which normal base pairing is never observed. Hence, ATP hydrolysis appears necessary for the RecA filament to be able to promote true renaturation. The renaturation reaction is found more effective when one of the complementary DNA strands is bound in the primary RecA DNA-binding position and the other is added as the third strand, but the reaction can also occur between DNA strands in any combination of binding positions in the RecA filament. This observation suggests the importance of the third DNA-binding position of the RecA filament. Renaturation between DNA strands in the other two combinations of binding positions is speculated to have a role in aborting the strand-exchange reaction when the strands are insufficiently complementary.
为了深入了解RecA促进互补多核苷酸之间碱基配对的机制，研究了在ATP（和ATP再生系统）存在下RecA与聚（dA）和聚（dT）的复合物形成。使用共价连接到聚（dA）不到1%的腺嘌呤碱基上的荧光探针苯并芘二环氧物（BPDE）跟踪反应。BPDE对其环境敏感，已发现它可用于检测DNA链之间的相互作用，在RecA细丝的三个结合位置中，在5'-O-3-硫代三磷酸腺苷（ATP[S]）存在的情况下[维通，P.，诺登，B.和高桥，M.（1994年）《生物化学杂志》269，5799-5803]。在ATP存在下形成的RecA:BPDE-聚（dA）的发射光谱与用ATP[S]观察到的光谱相似，支持复合物的相似结构。然而，荧光各向异性显著降低，表明在ATP水解存在下DNA具有更高的自由度。在ATP存在下，向预先形成的RecA:BPDE-聚（dA）细丝中加入互补链聚（dT）时，荧光强度缓慢降低，并观察到与沃森-克里克碱基配对一致的发射谱变化。这与ATP[S]的情况形成对比，在ATP[S]中从未观察到正常碱基配对。因此，ATP水解似乎是RecA细丝能够促进真正复性所必需的。当一条互补DNA链结合在RecA的主要DNA结合位置，另一条作为第三条链添加时，复性反应更有效，但反应也可以在RecA细丝中任何结合位置组合的DNA链之间发生。这一观察结果表明了RecA细丝的第三个DNA结合位置的重要性。推测在另外两种结合位置组合的DNA链之间的复性在链互补性不足时在中止链交换反应中起作用。
