Schutte B C, Cox M M
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin--Madison 53706.
Biochemistry. 1987 Sep 8;26(18):5616-25. doi: 10.1021/bi00392a006.
As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.
作为DNA链交换的第一步，RecA蛋白在单链DNA（ssDNA）上形成丝状复合物，该复合物含有化学计量（每四个核苷酸一个RecA单体）的RecA蛋白。该复合物中的RecA蛋白单体以25 min⁻¹的周转数水解ATP。引入线性同源双链DNA以启动链交换时，ATP水解速率下降33%。速率下降在不到2分钟内完成，然后在链交换反应期间及之后ATP水解速率保持恒定。这种下降完全取决于双链DNA中的同源性。此外，下降幅度与线性双链DNA中同源区域的长度呈线性相关。配对在拓扑上限制于平行排列连接的线性DNA底物也遵循这种关系。综上所述，这些特性表明，在DNA链交换反应的早期就能检测到进入的双链DNA中所有可用的同源性，线性双链DNA在其整个长度上与复合物中的同源ssDNA平行排列配对。结果表明平行排列连接可以延伸数千个碱基对。我们在其他地方指出[普格，B.F.，&考克斯，M.M.（1987b）《生物化学杂志》262，1337 - 1343]，这种双链对DNase消化的抗性获得过程要慢得多（30分钟），这与链交换的进程平行。这些结果共同表明双链DNA与ssDNA配对，但仍位于核蛋白丝之外。最后，结果也支持ATP水解发生在整个RecA核蛋白丝中的观点。
