Katz Francine S, Bryant Floyd R
Department of Biochemistry, The Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland 21205.
J Biol Chem. 2003 Sep 19;278(38):35889-96. doi: 10.1074/jbc.M305470200. Epub 2003 Jul 3.
The RecA protein from Escherichia coli promotes an ATP-dependent three-strand exchange reaction between a circular single-stranded DNA (ssDNA) and a homologous linear double-stranded (dsDNA). We have now found that under certain conditions, the RecA protein is also able to promote the three-strand exchange reaction using the structurally related nucleoside triphosphate, ITP, as the nucleotide cofactor. However, although both reactions are stimulated by single-stranded DNA-binding (SSB) protein, the ITP-dependent reaction differs from the ATP-dependent reaction in that it is observed only at low SSB protein concentrations, whereas the ATP-dependent reaction proceeds efficiently even at high SSB protein concentrations. Moreover, the circular ssDNA-dependent ITP hydrolysis activity of the RecA protein is strongly inhibited by SSB protein (suggesting that SSB protein displaces RecA protein from ssDNA when ITP is present), whereas the ATP hydrolysis activity is uninhibited even at high SSB protein concentrations (because RecA protein is resistant to displacement by SSB protein when ATP is present). These results suggest that SSB protein does not stimulate the ITP-dependent strand exchange reaction presynaptically (by facilitating the binding of RecA protein to the circular ssDNA substrate) but may act postsynaptically (by binding to the displaced strand that is generated when the circular ssDNA invades the linear dsDNA substrate). Interestingly, the mechanistic characteristics of the ITP-dependent strand exchange reaction of the E. coli RecA protein are similar to those of the ATP-dependent strand exchange reaction of the RecA protein from Streptococcus pneumoniae. These findings are discussed in terms of the relationship between the dynamic state of the RecA-ssDNA filament and the mechanism of the SSB protein-stimulated three-strand exchange reaction.
来自大肠杆菌的RecA蛋白能促进环状单链DNA（ssDNA）与同源线性双链（dsDNA）之间依赖ATP的三链交换反应。我们现在发现，在某些条件下，RecA蛋白还能够使用结构相关的核苷三磷酸ITP作为核苷酸辅因子来促进三链交换反应。然而，尽管这两种反应都受到单链DNA结合（SSB）蛋白的刺激，但依赖ITP的反应与依赖ATP的反应不同，前者仅在低SSB蛋白浓度下才能观察到，而依赖ATP的反应即使在高SSB蛋白浓度下也能高效进行。此外，RecA蛋白的环状ssDNA依赖的ITP水解活性受到SSB蛋白的强烈抑制（这表明当存在ITP时，SSB蛋白会将RecA蛋白从ssDNA上置换下来），而即使在高SSB蛋白浓度下，ATP水解活性也不受抑制（因为当存在ATP时，RecA蛋白对被SSB蛋白置换具有抗性）。这些结果表明，SSB蛋白不是在突触前刺激依赖ITP的链交换反应（通过促进RecA蛋白与环状ssDNA底物的结合），而是可能在突触后起作用（通过结合环状ssDNA侵入线性dsDNA底物时产生的被置换链）。有趣的是，大肠杆菌RecA蛋白依赖ITP的链交换反应的机制特征与肺炎链球菌RecA蛋白依赖ATP的链交换反应的机制特征相似。我们从RecA-ssDNA细丝的动态状态与SSB蛋白刺激的三链交换反应机制之间的关系方面对这些发现进行了讨论。
