SV40 T Antigen Structure and Helicase Mechanisms

SV40 T 抗原结构和解旋酶机制

• 批准号: 6771292
• 负责人: XIAOJIANG S CHEN
• 金额: $ 1.44万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6771292
• 关键词: DNA replication; DNA replication origin; adenosine triphosphate; conformation; crystallization; enzyme activity; enzyme mechanism; helicase; intermolecular interaction; oncoproteins; protein protein interaction; protein purification; protein structure function; simian virus 40; site directed mutagenesis; tumor antigens; virus antigen; virus genetics; virus protein; virus replication

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.

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。它还通过在起点周围组装成双六聚体，在病毒 DNA 复制中发挥重要作用，该双六聚体不仅充当解旋酶以打开起点并解开分叉 DNA，而且还充当招募必需的细胞复制蛋白的平台。例如 RPA。我们的目标是通过研究各种寡聚和构象状态的 LT 结构，了解这些不同生物过程中 LT 功能的结构基础。我们在 LT 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 片段结晶方面的最新进展建立在四个具体目标之上。 (i) 将确定包含 N 末端结构域的较大 LT 的晶体结构以及具有替代方案和潜在问题的逻辑方法。 审阅者认为这是一个经过高度改进的应用程序，解决了重要的问题。 这些目标的成功不仅将提供有关 SV40 T 抗原的新的、令人兴奋的信息，而且还将提供有关解旋酶和 DNA 复制起始的一般信息。 该应用程序的其他优势包括训练有素且生产力高的调查员、环境和优秀的合作者。 该应用程序的唯一弱点包括在某些情况下缺乏实验细节以及晶体学数据如何解决机制的清晰描述。 审阅者认为，与整个应用程序的优势相比，这些都是次要的。 综上所述，研究组对本次申请的热情很高。 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 SV40 LT 的理解和真核 DNA 复制领域产生重大影响。