Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA.
Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA.
复制标题
突触前丝的组装。
DOI：
10.1016/0022-2836(88)90442-1
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发表时间：
1988
影响因子：
5.6
通讯作者：
Griffith,JD
中科院分区：
文献类型：
--
作者：
Thresher,RJ;Christiansen,G;Griffith,JD
我们先前已经表明，SSB蛋白促进RecA蛋白到单链DNA（ssDNA）上的组装发生在三个步骤中：（1）SSB蛋白与ssDNA的快速结合，（2）RecA蛋白在该模板上成核，以及（3）额外的RecA蛋白的协同聚合以产生突触前纤维。在这里，电子显微镜已被用来进一步探索这个组装过程的参数。突触前纤维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（12 mm-Mg 2+存在时>3 mm）和相对低浓度的SSB蛋白（每18个核苷酸1个单体）。当SSB蛋白被添加到每九个核苷酸一个单体时，组装被抑制三倍。这些影响似乎是在成核步骤施加。成核后，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蛋白的浓度负相关。结合蔗糖沉降和电子显微镜分析确定，SSB蛋白从ssDNA在RecA蛋白组装被取代。
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 (>3 mmin 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.