Thresher R J, Christiansen G, Griffith J D
Lineberger Cancer Research Center, Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27514.
J Mol Biol. 1988 May 5;201(1):101-13. doi: 10.1016/0022-2836(88)90442-1.
We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.
我们之前已经表明，在单链结合蛋白（SSB）促进下，RecA蛋白组装到单链DNA（ssDNA）上分三步进行：（1）SSB蛋白与ssDNA快速结合；（2）RecA蛋白在该模板上成核；（3）额外的RecA蛋白协同聚合以产生突触前细丝。在此，电子显微镜已被用于进一步探究该组装过程的参数。突触前细丝形成的最佳程度要求每三个核苷酸至少有一个RecA蛋白单体、高浓度的ATP（在存在12 mM - Mg2+时大于3 mM）以及相对低浓度的SSB蛋白（每18个核苷酸1个单体）。当将SSB蛋白添加至每九个核苷酸一个单体时，组装受到三倍的抑制。这些效应似乎在成核步骤发挥作用。成核之后，RecA蛋白以每分钟250至900个RecA蛋白单体的净速率组装到ssDNA上，该速率与SSB蛋白的浓度呈负相关。蔗糖沉降和电子显微镜联合分析证实，在RecA蛋白组装过程中，SSB蛋白从ssDNA上被置换下来。
