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复制起始和终止调控中涉及的分子机制。已经发现各种应激条件激活细胞应答机制，其在不同水平抑制DNA复制的起始。从而降低了遗传信息受损的可能性，并将细胞进入致癌途径的风险降至最低。我们已经观察到，人类核仁素参与至少两个途径，以抑制DNA复制，但机制仍然不明确。沿着潜在的困难固有的压力条件下，细胞也有进化的设备（复制叉障碍; RFBs），以减少潜在的损害所造成的运动复制叉通过关键的基因组regions.In这项授权，我们首先提出的特征核仁素与人类复制蛋白A（hRPA）的相互作用。将鉴定对于与hRPA的物理相互作用至关重要的核仁素结构域，并表征各种强制降解条件诱导hRPA-核仁素复合物的能力。我们将在体外表征核仁素和hRPA磷酸化对复合物形成的影响。其次，我们描述了人p53和核仁素之间的相互作用。将确定p53和核仁素在体外和体内形成复合物所必需的关键结构域。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的能力的影响。我们将确定核仁素重新定位的p53要求。第三，我们的特点核仁素磷酸化在体内的作用。将确定调节与hRPA相互作用的核仁素上的关键磷酸化位点。将确定突变这些位点对体内各种核仁素活性的影响。最后，我们将描述TTF-I终止复制的机制。使用猿猴病毒40（SV 4 O）DNA复制系统，我们将分析在体外产生RFB的DNA要求。我们将描述RFB形成的最低蛋白质需求，并探测在停滞的复制叉发生的蛋白质-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.