STRUCTURE & FUNCTION OF THE SV40 T-AG DNA BINDING DOMAIN

结构

• 批准号: 2655005
• 负责人: WILLIAM W BACHOVCHIN
• 金额: $ 24.02万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2655005
• 关键词: DNA binding protein; DNA replication; DNA replication origin; chemical binding; chemical models; computer simulation; conformation; gel mobility shift assay; intermolecular interaction; molecular site; mutant; nuclear magnetic resonance spectroscopy; nucleic acid sequence; protein structure function; simian virus 40; site directed mutagenesis; structural biology; synthetic nucleotide; tumor antigens; virus genetics; virus protein

Initiation of DNA replication is a complicated process that is understood in only very broad terms. At present, the best eukaryotic model for studies of initiation of DNA replication is the Simian Virus 40 (SV4O) in vitro replication system. Initiation of SV4O replication requires a single viral protein termed T-antigen (T-ag). Critical roles played by T.ag during initiation of replication include site specific binding to the SV4O origin, catalysis of subsequent unwinding events and recruitment of additional proteins necessary for initiation of DNA synthesis. However, our understanding of these events is limited owing to a lack of structural information about T-ag or the T-ag DNA binding domain (T-ag-bd). The following specific aims are proposed to address these issues. I. To determine the structure of the DNA binding domain of SV4O T-antigen. II. To characterize the biochemical properties of the T-ag-bd, and of certain mutant forms of the T-ag-bd, and to examine the structures of those mutant forms of the T-ag-bd with interesting properties. III. To delineate the interaction of the T-ag-bd with DNA. The work proposed in this application is significant from a basic science standpoint because it will provide the first structure of a protein domain that recognizes an origin of replication, whether from a prokaryotic or eukaryotic source. Moreover, existing sequence data indicate the T-ag-bd is not related to other known DNA binding proteins, suggesting the structure may reveal a new protein structural motif. A knowledge of this structure, or of the structure of this domain complexed to DNA, will contribute significantly to advancing our understanding of replication by revealing the protein/DNA contacts that enable specific recognition and binding to an origin of replication. The health related significance of the work proposed stems from the fact that SV4O T-ag is very homologous to the T-ags encoded by the BK and JC viruses. These viruses induce a number of diseases in humans, including cancer. For example, JC virus induces progressive multifocal leukoencephalopathy, a disease present in many AIDS patients.

DNA复制的启动是一个复杂的过程 只是在非常宽泛的范围内。目前，最好的真核细胞模型 猴病毒40(SV4O)启动DNA复制的研究 体外复制系统。启动SV4O复制需要单个 称为T抗原(T-Ag)的病毒蛋白。T.ag扮演的关键角色 在复制启动期间包括与SV4O的特定位点结合 起因、催化随后的平仓事件和招募 启动DNA合成所需的额外蛋白质。然而， 我们对这些事件的了解有限，因为缺乏结构性的 有关T-ag或T-ag DNA结合域(T-ag-BD)的信息。这个 为解决这些问题，提出了以下具体目标。 1.确定SV40T抗原DNA结合区的结构。 II.鉴定T-Ag-BD的生化性质，以及 T-Ag-BD的某些突变形式，并检查其结构 T-Ag-BD的那些突变形式具有有趣的特性。 描述T-Ag-BD与DNA的相互作用。 本申请中提出的工作从基础科学角度具有重要意义。 因为它将提供蛋白质结构域的第一个结构 它识别复制的来源，无论是来自原核生物还是 真核来源。此外，现有的序列数据表明T-ag-BD 与其他已知的DNA结合蛋白无关，这表明 结构可能揭示了一个新的蛋白质结构基序。对这一点的了解 结构，或这个结构域的结构与DNA络合，将 通过以下方式显著促进我们对复制的理解 揭示蛋白质/DNA接触，使特定识别和 绑定到复制起点。 提出的工作与健康有关的意义源于这样一个事实 SV40T-AG与BK和JC编码的T-AGS有很高的同源性 病毒。这些病毒会导致人类患上多种疾病，包括 癌症。例如，JC病毒诱导进行性多灶性 白质脑病，一种存在于许多艾滋病患者中的疾病。