THE STRUCTURE AND FUNCTION OF THE SV40 TUMOR ANTIGEN

我们先前已经绘制并表征了DNA结合 SV70肿瘤（T）抗原的结构域。 我们的数据证明 残基131至371之间的区域作图对于 复制起点特异性DNA结合。 通过蛋白酶消化 实验中，我们还表明，一个“核心”区域映射， 从大约140到281是紧密相关的 DNA. 其他实验室的基因数据表明， 该核心区域内的序列从140映射到约260。 总之，这些发现定义了T抗原的最小区域， 这是与复制起点结合所必需的。 作为其中的一部分 建议的工作，我们计划确定核心内的序列 对DNA的正确结构至关重要的元素 结合区并与DNA接触。 这将 通过测试具有特定氨基酸的突变体的活性来完成 酸取代 除了DNA结合之外，SV40 T抗原还具有几种其他的免疫原性。 参与病毒DNA复制的活动。 这些 包括ATP酶、单链DNA结合活性、 解旋酶和来源特异性DNA解旋活性。 虽然 已经仔细研究了起始DNA结合和ATP酶结构域 地图，目前有很少的信息， 负责其他三项活动的区域。 我们 计划通过检查这些区域的体外活性， 各种胰蛋白酶片段。 最后，为了了解多功能 这种蛋白质的结构域与一个 另一个是在病毒DNA复制过程中，我们会产生各种各样的 检测缺失突变体对T抗原的影响 功能

We have previously mapped and characterized the DNA-binding domain of the SV70 tumor (T) antigen. Our data demonstrate that a region mapping between residues 131 to 371 is important for replication origin-specific DNA binding. By protease digestion experiments, we have also shown that a "core" region mapping from approximately 140 to 281 is tightly associated with the DNA. Genetic data from other labs indicate that the essential sequences within this core region map from 140 to about 260. Together, these findings define the minimal region of T antigen which is needed to bind to the replication origin. As part of this proposed work, we plan to identify the sequences within the core element that are essential for the proper structure of the DNA- binding region and for making contacts with the DNA. This will be done by testing the activity of mutants with specific amino acid substitutions. Beside DNA-binding, the SV40 T antigen has several other activities that are involved in virus DNA replication. These include an ATPase, a single-strand DNA-binding activity, a helicase and an origin-specific DNA unwinding activity. Although the origin DNA-binding and ATPase domains have been carefully mapped, there is presently very little information about the regions that are responsible for the other three activities. We plan to map these regions by examining the in vitro activity of various tryptic fragments. Finally, in order to understand how the multiple functional domains of this protein communicate and interact with one another during viral DNA replication, we will generate various deletion mutants and test the effect of the deletions on T antigen function.