MECHANISMS OF SV40 DNA REPLICATION

描述：本提案的长期目标是了解 真核细胞染色体DNA复制的基本机制， 确定这些机制在转化细胞中是如何被破坏的。 细胞的转化涉及对细胞内细胞的分化起始的失调。 染色体DNA复制 因此，他们的研究将有可能 提供有关真核DNA复制步骤的信息， 作为细胞内的调控点。 他们将使用猿猴病毒40（SV 40）作为体外模型系统， 真核生物DNA复制的机制。 首先，他们将隔离， 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， SV 40大肿瘤抗原（T抗原）是T抗原 以双六聚体而不是六聚体结合DNA。 的相互作用 具有合成复制泡状底物的T抗原双六聚体 (ie.,含有侧接双链体DNA的中心ssDNA区的底物） 将被化学和酶试剂探测。 他们将研究 T抗原双六聚体对长DNA分子解旋的作用， 并确定其他复制组件对稳定性的影响， T抗原双六聚体。 第三，他们将描述该机制 T抗原DNA解旋酶的DNA解旋。 他们将决定 DNA解旋酶步长和T抗原在DNA解旋过程中产生的DNA接触 通过使用核糖取代的DNA叉来解旋。 模型T 抗原在复制叉处环绕两条单链中的一条， 得到考验 第四，他们将研究DNA解旋反应介导的 hRPA在伪源底物上的表达。 他们之前发现hRPA可以结合 和含有结构特征（8 nt气泡）的变性DNA底物 在ATP依赖性T抗原-SV 40起源复合物中发现。 他们将 检查伪源序列和结构对hRPA结合的作用， 解旋这些基材。

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.