SV40 T Antigen Structure and Helicase Mechanisms

SV40大T抗原(LT)是一种具有多种生物学功能的病毒癌蛋白。它参与了细胞内 通过调节肿瘤抑制因子的活性进行转化。它在病毒感染中也扮演着重要的角色 通过围绕原点组装成双六聚体进行DNA复制，该双六聚体不仅具有解旋酶的功能 打开起源和解开分叉DNA，也是招募必要细胞的平台 复制蛋白，如RPA。我们的目标是了解这些函数中LT函数的结构基础 通过研究LT在不同寡聚体和构象状态下的结构，研究不同的生物过程。 四个具体的目标是建立在我们最近在LT和 含有较大C-末端部分(残基251-630)的LT片段的结晶。(I)水晶 包含N-末端结构域的较大LT的结构将被确定为了解如何不同 域之间相互作用。这些结构将揭示齐聚的特定相互作用 对DNA解旋酶活性很重要。这些结构还将提供对 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 LT与DNA在原点和复制分叉处的相互作用的研究将通过确定 含有LT片段和相应DNA底物的络合物的结构。(Iii) 在存在和不存在ATP及其类似物的情况下，将确定LT的结构，以便 了解三磷酸腺苷诱导构象转换的机制。这些结构将阐明 为ATP驱动的DNA解离过程提供了结构基础，并为 通过点突变研究LT的结构/功能关系。(Iv)最后，LT的复合体的结构 RPA将被确定为了解两种蛋白质之间的分子相互作用，这将 深入了解LT如何在原点和分叉DNA展开中与RPA协调。这项研究将 为理解细胞周期调控的一般分子机制提供有用的信息 真核细胞中的DNA复制过程，这两个过程都将对潜在的抗肿瘤药物的开发具有重要的价值 肿瘤病毒策略，以及人类对导致癌症的异常细胞周期的干预。

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.