Mechanisms of SV40 DNA Replication

描述（由申请人提供）：本提案的长期目标是了解参与哺乳动物DNA复制起始和终止调控的分子机制。已经发现各种应激条件可以激活细胞反应机制，从而在不同水平上抑制DNA复制的启动。遗传信息受损的可能性因此降低，细胞进入致癌途径的风险也降至最低。我们已经观察到人类核蛋白至少参与了两种抑制DNA复制的途径，但其机制仍不明确。除了应激条件下固有的潜在困难外，细胞还进化出了一些装置（复制叉屏障；rfb），以减少复制叉在关键基因组区域移动所造成的潜在损害。在本研究中，我们首次提出表征核仁蛋白与人复制蛋白A （hRPA）的相互作用。将确定与hRPA物理相互作用至关重要的核蛋白结构域，并表征各种应激条件诱导hRPA-核蛋白复合物的能力。我们将描述核仁蛋白和hRPA磷酸化对体外复合体形成的影响。其次，我们描述了人类p53和核蛋白之间的相互作用。将确定p53和核蛋白上的关键结构域，这些结构域是体外和体内复杂形成所必需的。我们将研究核蛋白对p53转录反激活参与细胞周期控制的基因的能力的影响。我们将确定p53对核蛋白再定位的要求。第三，我们描述了核蛋白磷酸化在体内的作用。将确定核蛋白上调节与hRPA相互作用的关键磷酸化位点。这些位点的突变对体内各种核蛋白活性的影响将被确定。最后，我们将描述ttf - 1终止复制的机制。利用猿猴病毒40 (sv40) DNA复制系统，我们将分析体外生成RFB所需的DNA。我们将描述RFB形成的最低蛋白质需求，并探索在停滞复制叉处发生的蛋白质- dna相互作用。

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to understand the molecular mechanisms involved in the regulation of the initiation and termination of mammalian DNA replication. Various stress conditions have been found to activate cellular response mechanisms that repress the initiation of DNA replication at various levels. The possibility of damage to the genetic information is thus reduced, and the risk that cells will enter the pathway of carcinogenesis is minimized. We have observed that human nucleolin is involved in at least two pathways that act to inhibit DNA replication, but the mechanisms remain ill-defined. Along with the potential difficulties inherent in stress conditions, cells also have evolved devices (replication fork barriers; RFBs) to reduce the potential for damage caused by the movement of replication forks through critical genomic regions.In this grant, we first propose to characterize the interaction of nucleolin with human replication protein A (hRPA). The nucleolin domains that are critical for physical interaction with hRPA will be identified, and the ability of various stress conditions to induce the hRPA-nucleolin complex will be characterized. We will characterize the effect of nucleolin and hRPA phosphorylation on complex formation in vitro. Second, we characterize the interaction between human p53 and nucleolin. The critical domains on p53 and nucleolin necessary for complex formation in vitro and in vivo will be identified. We will examine the effect of nucleolin on the ability of p53 to transcriptionally transactivate genes involved in cell-cycle control. We will determine the p53 requirements for nucleolin relocalization. Third, we characterize the role of nucleolin phosphorylation in vivo. The critical phosphorylation sites on nucleolin that modulate the interaction with hRPA will be identified. The effects of mutating these sites on various nucleolin activities in vivo will be determined. Lastly, we will characterize the mechanism of replication termination by TTF-l. Using the simian virus 40 (SV4O) DNA replication system, we will analyze the DNA requirements for generation of an RFB in vitro. We will characterize the minimal protein requirements for formation of the RFB, and probe the protein-DNA interactions occurring at a stalled replication fork.