Haruta Nami, Yu Xiong, Yang Shixin, Egelman Edward H, Cox Michael M
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2003 Dec 26;278(52):52710-23. doi: 10.1074/jbc.M308563200. Epub 2003 Oct 6.
The RecA proteins of Escherichia coli (Ec) and Deinococcus radiodurans (Dr) both promote a DNA strand exchange reaction involving two duplex DNAs. The four-strand exchange reaction promoted by the DrRecA protein is similar to that promoted by EcRecA, except that key parts of the reaction are inhibited by Ec single-stranded DNA-binding protein (SSB). In the absence of SSB, the initiation of strand exchange is greatly enhanced by dsDNA-ssDNA junctions at the ends of DNA gaps. This same trend is seen with the EcRecA protein. The results lead to an expansion of published hypotheses for the pathway for RecA-mediated DNA pairing, in which the slow first order step (observed in several studies) involves a structural transition to a state we designate P. The P state is identical to the state found when RecA is bound to double-stranded (ds) DNA. The structural state present when the RecA protein is bound to single-stranded (ss) DNA is designated A. The DNA pairing model in turn facilitates an articulation of three additional conclusions arising from the present work. 1) When a segment of a RecA filament bound to ssDNA is forced into the P state (as RecA bound to the ssDNA immediately adjacent to dsDNA-ssDNA junction), the segment becomes "pairing enhanced." 2) The unusual DNA pairing properties of the D. radiodurans RecA protein can be explained by postulating this protein has a more stringent requirement to initiate DNA strand exchange from the P state. 3) RecA filaments bound to dsDNA (P state) have directly observable structural changes relative to RecA filaments bound to ssDNA (A state), involving the C-terminal domain.
大肠杆菌（Ec）和耐辐射球菌（Dr）的RecA蛋白均能促进涉及两条双链DNA的DNA链交换反应。DrRecA蛋白促进的四链交换反应与EcRecA促进的反应相似，只是该反应的关键部分会受到Ec单链DNA结合蛋白（SSB）的抑制。在没有SSB的情况下，DNA缺口末端的双链DNA-单链DNA连接会极大地增强链交换的起始。EcRecA蛋白也呈现出相同的趋势。这些结果导致已发表的关于RecA介导的DNA配对途径的假设得到扩展，其中缓慢的一级步骤（在多项研究中观察到）涉及向我们指定为P状态的结构转变。P状态与RecA与双链（ds）DNA结合时发现的状态相同。RecA蛋白与单链（ss）DNA结合时存在的结构状态指定为A。DNA配对模型反过来有助于阐明这项工作得出的另外三个结论。1）当与单链DNA结合的RecA丝段被迫进入P状态时（如RecA与紧邻双链DNA-单链DNA连接的单链DNA结合），该段变得“配对增强”。2）耐辐射球菌RecA蛋白异常的DNA配对特性可以通过假设该蛋白从P状态启动DNA链交换有更严格的要求来解释。3）与双链DNA结合的RecA丝（P状态）相对于与单链DNA结合的RecA丝（A状态）具有直接可观察到的结构变化，涉及C末端结构域。
