MECHANISMS OF SV40 DNA REPLICATION

SV40 DNA 复制机制

• 批准号: 6373202
• 负责人: JAMES A. BOROWIEC
• 金额: $ 39.15万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6373202
• 关键词: DNA binding protein; DNA replication; DNA replication origin; casein kinase; enzyme activity; helicase; phosphorylation; simian virus 40; tumor antigens; virus DNA; virus antigen; virus replication

DESCRIPTION: The long term goals of this proposal are to understand the fundamental mechanisms of eukaryotic chromosomal DNA replication and to determine how these mechanisms are disrupted in transformed cells. Transformation of cells involves deregulation of the initiation of chromosomal DNA replication. Therefore, their research will potentially give information concerning the steps of eukaryotic DNA replication that act as regulatory control points inside the cell. They will use simian virus 40 (SV40) as a model system in vitro to explore the mechanism of eukaryotic DNA replication. First, they will isolate and characterize human proteins that physically interact with human replication protein A (hRPA). Second, they have found that the DNA helicase activity of the SV40 large tumor antigen (T antigen) is 15-fold greater when T antigen binds DNA as a double hexamer rather than as a hexamer. The interaction of the T antigen double hexamer with synthetic replication bubble substrates (ie., substrates that contain a central ssDNA region flanked by duplex DNA) will be probed by chemical and enzymatic reagents. They will examine the role of the T antigen double hexamer on unwinding of lengthy DNA molecules, and determine the effect of other replication components on the stability of the T antigen double hexamer. Third, they will characterize the mechanism of DNA unwinding for the T antigen DNA helicase. They will determine the DNA helicase step size and the DNA contacts that T antigen makes during DNA unwinding through the use of ribose-substituted DNA forks. The model that T antigen encircles one of the two single-strands at a replication fork will be tested. Fourth, they will examine the DNA unwinding reaction mediated by hRPA on pseudo-origin substrates. They found previously that hRPA can bind and denature DNA substrates containing a structural feature (an 8 nt bubble) found within the ATP-dependent T antigen-SV40 origin complex. They will examine the role of pseudo-origin sequence and structure on hRPA binding and unwinding of these substrates.

描述：该提案的长期目标是了解 真核生物染色体DNA复制的基本机制 确定转化细胞中这些机制是如何被破坏的。 细胞的转化涉及到启动的失调 染色体DNA复制。 因此，他们的研究可能会 提供有关真核 DNA 复制步骤的信息 作为细胞内的调节控制点。 他们将使用猿猴病毒40（SV40）作为体外模型系统来探索 真核生物DNA复制机制。 首先，他们将隔离并 表征与人类复制物理相互作用的人类蛋白质 蛋白 A (hRPA)。 其次，他们发现 DNA 解旋酶活性 SV40 大肿瘤抗原（T 抗原）比 T 抗原大 15 倍 以双六聚体而不是六聚体的形式结合 DNA。 的相互作用 具有合成复制泡底物的 T 抗原双六聚体 （即，含有中心 ssDNA 区域且两侧为双链 DNA 的底物） 将通过化学和酶试剂进行探测。 他们将检查 T抗原双六聚体在长DNA分子解旋中的作用， 并确定其他复制成分对稳定性的影响 T抗原双六聚体。 第三，他们将描述机制 T 抗原 DNA 解旋酶的 DNA 解旋过程。 他们将确定 DNA 解旋酶步长和 T 抗原在 DNA 过程中形成的 DNA 接触 通过使用核糖取代的 DNA 叉进行解旋。 T 的型号 抗原在复制叉处环绕两条单链之一 被测试。 第四，他们将检查由以下因素介导的 DNA 解旋反应： 假源底物上的 hRPA。 他们之前发现 hRPA 可以结合 并使含有结构特征的 DNA 底物变性（8 nt 气泡） 发现于 ATP 依赖性 T 抗原-SV40 起源复合物中。 他们会 检查假源序列和结构对 hRPA 结合的作用 这些基材的退绕。