MECHANISMS OF EUKARYOTIC DNA REPLICATION

真核 DNA 复制机制

• 批准号: 2065341
• 负责人: JAMES A. BOROWIEC
• 金额: $ 29.13万
• 依托单位: NEW YORK UNIVERSITY
• 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 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2065341
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA replication; DNA replication origin; RNA; eukaryote; helicase; host organism interaction; nucleic acid sequence; nucleic acid structure; protein structure function; simian virus 40; tumor antigens; virus DNA; virus replication

DESCRIPTION (Adapted from the applicant's abstract): 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. Since chromosomal DNA replicationis deregulated in transformed cells, this proposal may provide information on steps of eukaryotic DNA replication that act as regulatory checkpoints for the cell. Simian Virus 40 (SV40) will be used as a model system in vitro to explore the mechanisms of eukaryotic DNA replication. The investigator will examine how the SV40 origin of replication (ori) becomes denatured during the initiation phase of viral DNA replication. Critical contacts on ori that are used by SV40 large T antigen and human single-stranded DNA-binding protein (hRPA) to denature ori will be determined. The ori region contains three essential domains, the early palindrome, a 17bp AT region, and four pentanucleotides (GAGGC) to which T antigen directly binds. A second aim is to characterize the function of these essential domains within the ori region. Populations of SV40 origins will be prepared in which the early palindrome and AT region are replaced by random sequences, followed by selection for functional origins using either viral DNA replication(in vivo or in vitro) or the T-antigen dependent DNA unwinding reaction.Thirdly, the mechanism by which the DNA helicase activity of T antigen unwinds DNA will be examined. The unwinding of chemically-modified or ribose-substituted DNA forks by T antigen will be examined to determine the DNA helicase step size. The essential features of the DNA helicase mechanism will be examined bycharacterizing the binding of the T antigen RNA helicase to RNA forks as a comparative tool.Finally, the role of hRPA during SV40 DNA replication will be examined. Studies in the investigator's laboratory and in other labs have found that hRPA can form two markedly different complexes with single-stranded DNA. The interaction of hRPA in these complexes with modified single stranded DNA binding substrates will be individually characterized.

描述（改编自申请人的摘要）：长期目标 该提案的目的是了解的基本机制 真核生物染色体 DNA 复制并确定这些 转化细胞中的机制被破坏。由于染色体 DNA 复制在转化细胞中失调，该提议 可能提供有关真核 DNA 复制步骤的信息 作为细胞的监管检查点。 猿猴病毒 40 (SV40) 将用作体外模型系统 探索真核生物DNA复制的机制。 调查员 将检查 SV40 复制起点 (ori) 如何变性 在病毒DNA复制的起始阶段。 批判的 SV40 大 T 抗原和人类使用的 ori 上的接触 单链 DNA 结合蛋白 (hRPA) 使 ori 变性 决定。 ori 区域包含三个重要结构域， 早期回文，一个 17bp AT 区域和四个五核苷酸 (GAGGC) T抗原直接结合。 第二个目标是 描述 ori 内这些基本域的功能 地区。 将制备 SV40 起源的群体，其中 早期回文和 AT 区域被随机序列替换， 然后使用病毒 DNA 选择功能起源 复制（体内或体外）或 T 抗原依赖性 DNA 解旋反应。第三，DNA解旋酶的机制 将检查 T 抗原解旋 DNA 的活性。 的放松 T 抗原化学修饰或核糖取代的 DNA 叉将 进行检查以确定 DNA 解旋酶步长。 必不可少的 将检查 DNA 解旋酶机制的特征 通过表征 T 抗原 RNA 解旋酶与 RNA 叉的结合 作为比较工具。最后，hRPA 在 SV40 DNA 过程中的作用 将检查复制情况。 研究人员的研究 实验室和其他实验室发现 hRPA 可以形成两种显着的 不同的单链DNA复合物。 hRPA 的相互作用 在这些具有修饰的单链 DNA 结合底物的复合物中 将被单独表征。