SV40 T Antigen Structure and Helicase Mechanisms

描述（由申请方提供）：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。它通过调节肿瘤抑制因子的活性参与细胞转化。它还在病毒DNA复制中发挥重要作用，通过围绕起点组装成双六聚体，不仅作为解旋酶打开起点并解开叉DNA，而且作为招募必需的细胞复制蛋白（如RPA）的平台。我们的目标是了解LT功能在这些不同的生物过程中的结构基础，通过研究LT结构在其各种寡聚体和构象状态。四个具体的目标是建立在我们最近的进展，在生物化学/蛋白质化学的LT和结晶的LT片段含有较大的C-末端部分（残基251-630）。(i)含有N-末端结构域的较大LT的晶体结构将被确定为具有替代方案和潜在问题的逻辑方法。 审查人员认为，这是一个高度改进的应用程序，解决了重要问题。 这些目标的成功不仅将提供关于SV 40 T抗原的新的和令人兴奋的信息，而且还将提供关于解旋酶和DNA复制起始的一般信息。 该应用程序的其他优势包括受过良好培训且富有成效的调查员，环境和优秀的合作者。 在某些情况下，应用程序中唯一的弱点包括缺乏实验细节，以及晶体学数据如何解决机制的清晰描述。 审查人员认为，与整个应用程序的优点相比，这些都是次要的。 总之，研究组对这一申请热情很高。 他们认为，根据初步数据，成功的可能性很高，问题很重要，这项研究的结果将对理解SV 40 LT和真核DNA复制领域产生重大影响。

DESCRIPTION (provided by applicant): 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 leaas well as a logical approach with alternatives and potential problems. The reviewers felt that this was a highly improved application that addressed important questions. Success of these aims will not only provide new and exciting information about SV40 T antigen, but also about helicases and DNA replication initiation, in general. Further strengths of this application include the investigator, who is well trained and has been highly productive, the environment, and the excellent collaborators. The only weaknesses in the application include, in some cases, a lack of experimental detail, and a clear description of how the crystallographic data will address mechanism. The reviewers felt that these were minor compared to the strengths of the overall application. In summary, the study section was highly enthusiastic about this application. They felt that based on the preliminary data there was a high likelihood of success, the questions were significant, and the results from this study will have a high impact on both the understanding of SV40 LT and the field of eukaryotic DNA replication.