Viral DNA Replication and Late Transcription

病毒 DNA 复制和后期转录

• 批准号: 6671993
• 负责人: Linda A. Guarino
• 金额: $ 7.05万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6671993
• 关键词: Baculoviridae; DNA directed RNA polymerase; DNA replication; DNA replication origin; SDS polyacrylamide gel electrophoresis; enhancer binding protein; gel mobility shift assay; genetic transcription; matrix assisted laser desorption ionization; protein binding; protein protein interaction; protein purification; protein structure function; transcription factor; virus DNA

DESCRIPTION (provided by applicant): Transcription of baculovirus late genes is absolutely dependent upon viral DNA replication, which is believed to create or unmask a cis-acting signal in the DNA template. In vitro analyses with the viral-encoded RNA polymerase suggests that this signal is the replication fork, and that proteins bound to the fork enhance late transcription. The goals of this proposal are to identify the proteins that enhance late transcription and to define their mechanism of action. Enhancers will be purified using a complementation assay, which consists of purified viral RNA polymerase, a DNA template containing a replication fork, and chromatography fractions. Forked templates are preferentially enhanced suggesting that proteins bind specifically to replication forks. Therefore, the DNA binding specificity of the enhancers will be determined by electrophoretic mobility shift assays using linear, nicked, and forked probes. The interactions between the enhancers, RNA polymerase, forks, and baculovirus promoters will be analyzed by protein cross-linking and DNase I footprinting assays. The mechanism of enhancer function will be further characterized by permanganate probing and abortive initiation assays. Finally, to determine whether the replication fork is a position and orientation-independent element, different circular and linear templates, which contain replication forks upstream, downstream, and on both strands of DNA will be constructed. Comparison of the transcription activities of these templates will help to determine whether the replication fork enhancers function similarly to classical transcription enhancers. These experiments should provide an explanation for the dependence of late transcription on DNA replication. The enhancer factors act preferentially on forked DNA templates, and the primary intracellular mechanism of generating forked templates is through DNA replication. If the viral RNA polymerase is dependent on forked structures for enhanced transcription, then late transcription cannot occur in the absence of DNA replication, even though RNA polymerase and the late transcription factors are encoded by early genes. Knowledge gained from these experiments should be relevant to other viral systems and will provide a better understanding of the complex interactions between transcription and replication machinery in eukaryotic cells.

描述（由申请人提供）：杆状病毒后期基因的转录绝对取决于病毒DNA复制，据信这在DNA模板中创建或揭示了顺式作用信号。 用病毒编码的RNA聚合酶进行体外分析表明，该信号是复制叉，并且与叉子结合的蛋白质增强了晚期转录。 该提案的目标是确定增强晚期转录并定义其作用机理的蛋白质。 增强剂将使用互补测定法进行纯化，该互补测定包括纯化的病毒RNA聚合酶，该聚合酶是包含复制叉和色谱分数的DNA模板。 分叉模板优先增强，表明蛋白质专门与复制叉结合。 因此，增强子的DNA结合特异性将由使用线性，划痕和分叉探针的电泳迁移率转移测定确定。 增强剂，RNA聚合酶，叉子和杆状病毒启动子之间的相互作用将通过蛋白质交联和DNase I足迹测定法分析。 增强子功能的机制将进一步以锰酸盐的探测和流产性起始分析为特征。 最后，确定复制叉是否是位置和方向无关的元素，将构建包含复制叉上游，下游和两个DNA链的不同圆形和线性模板。 这些模板的转录活性的比较将有助于确定复制叉增强剂是否与经典转录增强剂相似。 这些实验应为晚期转录对DNA复制的依赖性提供解释。 增强子因子优先在分叉的DNA模板上起作用，生成分叉模板的主要细胞内机制是通过DNA复制。 如果病毒RNA聚合酶取决于分叉结构以增强转录，那么即使RNA聚合酶和晚期转录因子由早期基因编码，也无法在没有DNA复制的情况下进行晚期转录。 从这些实验中获得的知识应与其他病毒系统有关，并将更好地理解真核细胞中转录和复制机制之间的复杂相互作用。