MECHANISMS OF EUKARYOTIC DNA REPLICATION

描述（改编自申请人的摘要）：长期目标 这一建议的目的是了解的基本机制 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。由于染色体 DNA复制在转化细胞中被解除管制， 可以提供真核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。 研究者 将研究SV40复制起点（ori）如何变性 在病毒DNA复制的起始阶段。 关键 SV40大T抗原和人使用的ori上的接触 单链DNA结合蛋白（hRPA）使ori变性， 测定 ori区域包含三个基本结构域， 早期回文序列，一个17bp的AT区和四个五核苷酸 T抗原直接结合的GAGGC。 第二个目标是 描述这些重要结构域在ori中的功能 地区 将制备SV40来源的群体，其中 早期回文和AT区被随机序列取代， 随后使用病毒DNA或DNA片段选择功能性来源， 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 检测T抗原解旋DNA的活性。 的退绕 被T抗原化学修饰或核糖取代的DNA叉将 以确定DNA解旋酶步长。 的基本 DNA解旋酶机制的特点将被检查 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中的作用， 将审查复制情况。 研究者的研究 实验室和其他实验室已经发现，hRPA可以形成两个显着 与单链DNA形成不同的复合物。 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， 将被单独描述。

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.