MECHANISMS OF EUKARYOTIC DNA REPLICATION

描述(改编自申请人的摘要)：长期目标 这一建议的基本原理是理解 真核细胞染色体DNA复制，并确定这些 在转化的细胞中，机制被破坏。从染色体开始 DNA复制在转化细胞中被解除调控，这一提议 可以提供有关真核DNA复制的步骤的信息 作为细胞的监管检查点。 猴病毒40(SV40)将作为体外模型系统用于 探索真核细胞DNA复制的机制。调查员 将研究SV40复制起点(ORI)是如何变性的 在病毒DNA复制的起始阶段。批判性 SV40大T抗原与人ORI的接触 单链DNA结合蛋白(HrpA)将被 下定决心。ORI区域包含三个基本域，即 早期回文，17bp的AT区和4个五核苷酸 (GAGGC)与T抗原直接结合。第二个目标是 描述ORI中这些基本领域的功能 区域。将准备SV40起源的种群，其中 将早期回文和AT区替换为随机序列， 然后使用两种病毒DNA中的一种选择功能来源 复制(体内或体外)或T抗原依赖的DNA 解离反应。第三，DNA解旋酶 T抗原解链DNA的活性将被检测。的解体 T抗原化学修饰或核糖取代的DNA叉会 被检查以确定DNA解旋酶的步长。最重要的 我们将研究dna解旋酶机制的特点。 通过表征T抗原RNA解旋酶与RNA叉的结合 作为一种比较工具。最后，HrpA在SV40 DNA中的作用 我们将检查复制。在调查员的研究 实验室和其他实验室已经发现，HrpA可以明显形成两种 与单链DNA形成不同的复合体。HrpA的相互作用 在这些具有修饰的单链DNA结合底物的复合体中 将被单独定性。

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.