Mechanisms of SV40 DNA Replication

描述(由申请人提供)：本提案的长期目标是了解哺乳动物DNA复制的启动和终止的调控的分子机制。不同的应激条件可以激活细胞反应机制，在不同水平上抑制DNA复制的启动。因此，遗传信息受损的可能性降低，细胞进入致癌途径的风险也降至最低。我们观察到，人类核仁素至少参与了两条抑制DNA复制的途径，但其机制仍不明确。除了在应激条件下固有的潜在困难外，细胞还进化了一些装置(复制叉屏障；RFB)来减少复制叉通过关键基因组区域造成的损害的可能性。在这项授权中，我们首先提出了核仁素与人类复制蛋白A(HrpA)相互作用的特征。将确定对与HrpA物理相互作用至关重要的核仁域，并将表征各种应力条件诱导HrpA-核素复合体的能力。我们将在体外研究核仁素和HrpA磷酸化对复合体形成的影响。其次，我们描述了人类P53和核仁素之间的相互作用。将确定在体外和体内形成复合体所必需的P53和核仁上的关键结构域。我们将研究核仁素对P53转录反式激活参与细胞周期控制的基因能力的影响。我们将确定核仁重定位所需的P53。第三，我们研究了核仁磷酸化在体内的作用。核仁上调节与HrpA相互作用的关键磷酸化位点将被识别。突变这些位点对体内各种核仁素活性的影响将被确定。最后，我们将对TTF-L终止复制的机制进行表征。利用猴病毒40(SV4O)DNA复制系统，我们将在体外分析产生RFB所需的DNA。我们将描述形成RFB的最低蛋白质需求，并探索在停滞的复制叉子上发生的蛋白质-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.