Ullsperger C J, Cox M M
Department of Biochemistry, University of Wisconsin-Madison 53706, USA.
Biochemistry. 1995 Aug 29;34(34):10859-66. doi: 10.1021/bi00034a019.
Following a DNA strand exchange reaction, RecA protein remains bound to the hybrid DNA product. DNA strand exchange reactions were carried out under optimal conditions in the presence of both RecA protein and SSB protein. As monitored by a sensitive DNA underwinding assay, all of the RecA protein present in the RecA nucleoprotein filament that initiates the strand exchange reaction can be accounted for on the hybrid DNA. As shown elsewhere, the SSB is bound to the displaced single DNA strand. Previous studies showed that RecA protein will dissociate from dsDNA when ADP levels build up, or transfer from dsDNA to ssDNA when the latter is not bound by SSB. The present work (done with ATP regeneration and SSB) shows that efficient strand exchange occurs in the absence of a net dissociation or transfer of RecA monomers from the filament. Such a dissociation or transfer is therefore not a mechanistic requirement for DNA strand exchange. The results provide evidence against some models proposed for the DNA strand exchange mechanism.
在DNA链交换反应之后,RecA蛋白仍与杂交DNA产物结合。DNA链交换反应在RecA蛋白和SSB蛋白均存在的最佳条件下进行。通过灵敏的DNA解旋测定监测发现,引发链交换反应的RecA核蛋白丝中存在的所有RecA蛋白都可以在杂交DNA上找到。如其他地方所示,SSB与被置换的单链DNA结合。先前的研究表明,当ADP水平升高时,RecA蛋白会从双链DNA上解离,或者当单链DNA未被SSB结合时,RecA蛋白会从双链DNA转移到单链DNA上。目前的工作(在ATP再生和SSB存在的情况下完成)表明,在没有RecA单体从丝状体发生净解离或转移的情况下,高效的链交换仍会发生。因此,这种解离或转移不是DNA链交换的机制要求。这些结果为一些提出的DNA链交换机制模型提供了反证。