Analysis of the functional domains of a regulatory protein, RecA, using gene manipulation and X-ray crystallography.

由于其大小(38,000道尔顿)，RecA蛋白在体外表现出惊人的各种反应：RecA蛋白与单链DNA结合；(&lt；II&gt；)具有依赖DNA的ATPase；(&lt；III&gt；)催化互补单链DNA的复性；(&lt；iv&gt；)解开双链DNA；以及(&lt；v&gt；)裂解Lexa蛋白和&lt；lambda&gt；，P22，&lt；Phi&Gt；80噬菌体的阻断物。RecA蛋白可能有几个不同的位置直接参与这些不同的反应。为了阐明反应的分子机制，在蛋白质的氨基酸序列上定位这些反应位点将是有用的。因此，我们首先确定了我们新分离的一些或全部recA功能缺陷的recA突变体的突变位点，并根据突变蛋白和修饰的recA蛋白的生化和生物物理性质，预测了RecA蛋白的功能结构域。要了解这些反应是如何进行的，需要通过X射线结晶学来确定蛋白质的三维结构。我们正在努力获得质量良好的recA蛋白晶体，适用于详细的X射线结晶学分析。

For its size (38,000 daltons), the RecA protein exhibits a remarkable variety of reactions in vitro: RecA protein, ( <i> ) binds to a single-stranded DNA; ( <ii> ) has DNAdependent ATPase; ( <iii> ) catalyzes the renaturation of complementary single-strands of DNA; ( <iv> ) unwinds double stranded DNA; and ( <v> ) cleaves LexA protein and the repressors of <lambda> , P22, and <phi> 80 phage. RecA protein probably has several different sites involved directly in these various reactions. To elucidate the molecular mechanism of reactions, location of these reaction sites on the amino acid sequence of the protein would be useful. We therefore first determined the mutation sites of various recA mutants newly isolated by us which are deficient in some or all of the recA functions and, based on the biochemical and biophysical properties of the mutant proteins as well as those of the modified RecA protein, we predicted the functional domains of the RecA protein. To learn how the reactions are done will require determination of the protein's three dimensional structure by X-ray crystallography. We are making effort to obtain a good quality of recA protein crystals suitable for detailed X-ray crystallographic analysis.