SV40 T Antigen Structure and Helicase Mechanisms

描述(申请人提供)：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，它围绕起始点组装成一个双六聚体，不仅起到解开起始点和解开分叉DNA的解旋酶的作用，而且还作为招募必要的细胞复制蛋白的平台，如RPA。我们的目标是通过研究LT在不同的寡聚体和构象状态下的结构，来了解LT在这些不同生物过程中功能的结构基础。四个特定的目标是建立在我们最近在LT的生物化学/蛋白质化学和含有较大C-末端部分(残基251-630)的LT片段的结晶方面的进展。(I)将确定含有N-端结构域的较大LT的晶体结构以及替代方案和潜在问题的逻辑方法。审查者认为，这是一个解决了重要问题的高度改进的应用程序。这些目的的成功不仅将提供关于SV40T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复制启动的一般信息。这个应用程序的进一步优势包括训练有素且工作效率很高的调查员、环境和优秀的合作者。应用中唯一的缺点包括，在某些情况下，缺乏实验细节，以及对结晶学数据将如何解决机理的清楚描述。评审者认为，与整个应用程序的优势相比，这些都是次要的。总而言之，研究组对这一应用程序非常热情。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SV40 LT和真核DNA复制领域产生很大影响。

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.