Genetic and Immunological Analysis of E. coli recA Protein

大肠杆菌recA基因编码的蛋白质Eco Recap在DNA代谢的几个过程中起着至关重要的作用：(1)一般的基因重组；(2)DNA化学和辐射损伤的修复；(3)紫外线诱变；(4)DNA损伤后协调修复、复制和细胞分裂。在5个与大肠杆菌关系密切的细菌物种和9个远亲物种中发现了具有相似活性的recA类似物。目前已从真菌、昆虫、植物和哺乳动物细胞中分离出具有EcoRecAp活性的蛋白质。其中，对EcoRecAp的遗传学和酶学研究最为深入。然而，我们对EcoRecAp的结构以及该结构与酶活性的关系仍然知之甚少。标准的X射线结晶学技术已经应用于EcoRecAp，但由于蛋白质的灵活性，到目前为止还没有成功地揭示其结构。计算机分析已经检测到EcoRecAp的中心部分与一组同质蛋白质相似，称为平行AB蛋白质。这项提案的一半内容是对这个被称为核心的三维模型进行基因测试。这些测试的一部分是对几个物种的重复序列进行平均预测，这种方法成功地用于预测另一种平行AB蛋白质的三维结构。这项测试还包括检查改变特定氨基酸的预测结果。这些测试将通过一个类似于逐次逼近的过程，帮助改进模型，无论预测是确认的还是矛盾的。提案的另一半涉及EcoRecAp的氨基和羧基末端区域的结构和功能作用。这些区域的结构在核心模型中没有预测到。将研究两种单抗对EcoRecAp的影响。一个是MAb4B4，与氨基末端氨基酸结合；另一个是MAb156，与羧基末端氨基酸结合。这两种抗体的结合竞争可能揭示这些区域位于EcoRecAp结构中的距离有多近。每种抗体对EcoRecAp六个体外活性的影响可能揭示这些区域如何参与蛋白质的功能。

The protein encoded by the recA gene of E. coli, Eco RecAp, is of paramount importance in several processes of DNA metabolism: (1) general genetic recombination (2) repair of chemical and irradiation damage to DNA, (3) UV mutagenesis and (4) coordinating repair, replication, and cell division after DNA has been damaged. Analogues of recA with similar activity have been found in five bacterial species closely related to E. coli and nine distantly related species. Proteins with activities of EcoRecAp have now been isolated from fungal, insect, plant and mammalian cells. Of all these EcoRecAp has been most thoroughly studied genetically and enzymatically. Still, however, we have only limited insight into the structure of EcoRecAp and how that structure relates to enzymatic activity. The standard technique of x-ray crystallography has been applied to EcoRecAp but has so far been unsuccessful in revealing its structure because of the flexibility of the protein. Computer analysis has detected similarities of the central part of EcoRecAp with a homogeneous group of proteins, called parallel aB proteins. Half of this proposal consists of genetic tests of the three-dimensional model of this central part, which has been called the core. The tests consist in part of averaging predictions for RecAps from several species, a method used successfully to predict the three-dimensional structure of another parallel aB protein. The test also consist in part of examining the predicted results of altering specific amino acids. These tests will help refine the model, whether the predictions are confirmed or contradicted, by a process akin to successive approximations. The other half of the proposal concerns the structural and functional roles of the amino and carboxy terminal regions of EcoRecAp. These are the regions whose structure was not predicted in the model of the core. Two monoclonal antibodies will be studied for their effects on EcoRecAp. One, MAb4B4, binds to amino terminal amino acids; the other, MAb156, binds to carboxy terminal amino acids. Binding competition of the two antibodies may reveal how closely these regions are situated in the structure of EcoRecAp. Effects of each antibody on six in vitro activities of EcoRecAp may reveal how these regions participate in the functions of the protein.