MECHANISMS OF SV40 DNA REPLICATION

描述：这项建议的长期目标是了解 真核细胞染色体DNA复制的基本机制及其意义 确定这些机制在转化的细胞中是如何被破坏的。 细胞的转化涉及解除对启动 染色体DNA复制。因此，他们的研究可能会 提供有关真核DNA复制的步骤的信息 作为细胞内的调控控制点。 他们将使用猿猴病毒40(SV40)作为体外模型系统来探索 真核细胞DNA复制的机制。首先，他们将隔离和 鉴定物理上与人类复制相互作用的人类蛋白质 蛋白A(HrpA)。第二，他们发现DNA解旋酶的活性 SV40大肿瘤抗原(T抗原)是T抗原的15倍 以双六角体而不是六角体的形式结合DNA。的相互作用 具有合成复制气泡底物的T抗原双六聚体 (例如，含有中心单链DNA区域的底物，侧翼是双链DNA) 将通过化学和酶试剂进行探测。他们将审查 T抗原双六聚体在解离长DNA分子中的作用 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，他们将描述这种机制的特征 T抗原DNA解旋酶的DNA解离。他们将决定 DNA解旋酶步长与T抗原在DNA过程中的DNA接触 通过使用核糖取代的DNA叉来解开。T的模型 在复制叉处，抗原包围两条单链中的一条 接受测试。第四，他们将研究DNA解旋反应，该反应由 假起源底物上的HrpA。他们之前发现HrpA可以结合 和含有结构特征的变性DNA底物(8个核苷酸的气泡) 在依赖于ATP的T抗原-SV40起源复合体中发现。他们会 研究伪起源序列和结构在HrpA结合和 解开这些衬底。

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.