SV40 T Antigen Structure and Helicase Mechanisms

SV 40大T抗原（LT）是一种具有多种生物学功能的病毒癌蛋白。它参与了细胞 通过调节肿瘤抑制因子的活性来促进转化。它也在病毒中起着重要作用， DNA复制是围绕着起始点组装成一个双六聚体，它不仅作为解旋酶发挥作用， 打开起源，解开分叉DNA，但也作为一个平台， 复制蛋白，如RPA。我们的目标是了解LT功能的结构基础， 通过研究LT结构在其各种低聚体和构象状态下的不同生物过程。 四个具体的目标是建立在我们最近在LT的生物化学/蛋白质化学方面的进展和 含有较大C-末端部分（残基251-630）的LT片段结晶。(i)晶体 将确定含有N-末端结构域的较大LT的结构，以了解不同的结构域是如何形成的。 域之间相互作用。这些结构将揭示寡聚化的特定相互作用， 对DNA解旋酶活性很重要。这些结构也将提供深入了解的机制， LT如何通过细胞转化中的蛋白质-蛋白质相互作用调节肿瘤抑制因子。(ii)模式 将通过确定LT与DNA在起点和复制叉处的相互作用来研究 LT片段与相应DNA底物的复合物的结构。(iii)的 将测定存在和不存在ATP及其类似物时LT的结构， 了解ATP诱导构象转换的机制。这些结构将阐明 结构基础的ATP驱动的DNA解旋过程，并提供了指导的研究， LT结构/功能关系。(iv)最后，研究了LT配合物的结构 与RPA将确定了解两种蛋白质之间的分子相互作用，这将 深入了解LT如何与RPA在起源和分叉DNA解旋中协调。这项研究将 提供有助于理解细胞周期控制的一般分子机制的信息， 真核生物中DNA复制过程的研究，这两方面的研究对于开发抗肿瘤药物具有重要意义。 肿瘤病毒策略，以及对导致癌症的异常细胞周期的人为干预。

SV40 large T antigen (LT) is a viral oncoprotein with diverse biological functions. It is involved in cellular transformation through regulating the activities of tumor suppressors. It also plays an important role in viral DNA replication by assembling around the origin into a double hexamer that function not only as a helicase to open up the origin and unwind the fork DNA, but also as a platform for recruiting the essential cellular replication proteins, such as RPA. Our goals are to understand the structural basis of LT functions in these diverse biological processes by studying LT structures in their various oligomeric and conformational states. Four specific aims are built upon our recent progress in the biochemistry/protein chemistry of LT and the crystallization of a LT fragment containing the larger C-terminal portion (residues 251-630). (i) Crystal structures of larger LT containing the N-terminal domains will be determined to learn how the different domains interact with each other. The structures will reveal the specific interactions for oligomerization that are important for DNA helicase activity. These structures will also provide insight into the mechanisms of how LT regulates tumor suppressors through protein-protein interactions in cell transformation. (ii) The mode of interactions between LT and DNA at the origin and at the replication fork will be studied by determining the structures of the complexes containing LT fragments and the corresponding DNA substrates. (iii) The structures of LT in the presence and absence of ATP and its analogs will be determined in order to understand the mechanisms of the ATP induced conformational switch. These structures will elucidate the structural basis for the ATP-driven DNA-unwinding process, and provide a guide for the studies of the structure/function relationship of LT through site-mutagenesis. (iv) Finally, the structure of the complex of LT with RPA will be determined to understand the molecular interactions between the two proteins, which will provide insight into how LT coordinates with RPA in the origin and fork DNA unwinding. This research will provide information useful for understanding the general molecular mechanisms of cell cycle control and DNA replication process in eukaryotes, both of which will be valuable for the potential development of anti- tumor virus strategies, and for human intervention of abnormal cell cycles that lead to cancer.