STRUCTURE AND FUNCTION OF THE SV40 TUMOR ANTIGEN

我们之前已经定位并表征了DNA结合 SV70肿瘤(T)抗原的结构域。我们的数据表明 残基131到371之间的区域映射对于 复制原点特定的DNA结合。用酶消化法 实验中，我们还证明了一个“核心”区域的映射 从大约140到281与 DNA来自其他实验室的基因数据表明，基本的 该核心区内的序列从140到约260。 总之，这些发现定义了T抗原的最小区域 它是绑定到复制源所需的。作为这项工作的一部分 拟议的工作，我们计划识别核心内的序列 对DNA的正确结构至关重要的元素- 结合区域和与DNA的接触。这将是 是通过测试含有特定氨基酸的突变体的活性来完成的 酸替换。 除了DNA结合，SV40T抗原还有其他几种 参与病毒DNA复制的活动。这些 包括ATPase、单链DNA结合活性、 解旋酶和来源特定的DNA解离活性。虽然 起源的DNA结合结构域和ATPase结构域已经被仔细地 在地图上，目前关于 负责其他三项活动的地区。我们 计划通过检查体外活性来绘制这些区域的地图 各种胰酶碎片。 最后，为了理解多重功能是如何 这种蛋白质的结构域与一种 另外，在病毒DNA复制过程中，我们会产生各种 缺失突变并检测缺失对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.