Initiation of SV40 DNA Replication and Its Regulation

描述(申请人提供)：许多DNA肿瘤病毒编码“启动者” 与DNA复制的病毒起点特异结合的蛋白质。vt.在.的基础上 启动子与它们各自的起始点结合，组装成DNA解旋酶 能够融化双链DNA的物质。发起者还招募细胞 合成新生DNA所需的蛋白质。我们正试图理解 通过深入描述由猿猴病毒编码的启动子，这些过程 40，称为T抗原(T-Ag)。使用猿猴病毒40系统和Simple Band 转移实验，生物学中的一个基本问题将被解决：“如何 细胞周期机制是否控制病毒上启动子的组装 复制的起源？“最近对T-Ag衍生多肽的实验，其 苏氨酸124，ARE的磷酸化调节与DNA的结合能力 为这一过程提供了重要的见解。建议进行实验，以 确定类似的机制是否在T-ag环境中运行。相关 将考虑T-AG组装和监管的各个方面，例如设置 T-ag上与岩心起源中的侧翼序列相互作用的位点 是T-Ag结合所必需的。总的来说，这些研究将提供 这些信息可能会让我们解决DNA解开的机制。一旦 SV40起始处被解开，Pola-Primase复合体启动合成 新生的DNA链。到底是什么序列构成了起始点 因为POLα-底物酶复合体是另外一系列 实验。鉴于许多基本过程在整个过程中都是守恒的 进化，我们相信我们从这些研究中获得的信息 将与其他病毒来源的DNA复制的启动有关 复制的结果。此外，它们可能有助于我们理解细胞周期是如何 机械控制细胞起源的启动事件。考虑到 不受控制的DNA复制被认为是许多疾病的一个组成部分， 包括癌症在内，了解DNA复制是如何进行的非常重要 启动以及如何控制它。

DESCRIPTION (provided by applicant): Many DNA tumor viruses encode "initiators" proteins that site specifically bind to viral origins of DNA replication. Upon binding to their respective origins, the initiators assemble into DNA helicases that are capable of melting duplex DNA. Initiators also recruit cellular proteins required for synthesis of nascent DNA. We are attempting to understand these processes by characterizing in depth an initiator encoded by Simian Virus 40, termed T-antigen (T-ag). Using the Simian Virus 40 system and simple band shift experiments, a fundamental question in biology will be addressed: "how does the cell cycle machinery control the assembly of initiators on viral origins of replication?" Recent experiments with T-ag derived peptides, whose ability to bind to DNA is regulated by phosphorylation of Thr 124, are providing significant insights into this process. Experiments are proposed to determine if similar mechanisms are operating in the context of T-ag. Related aspects of T-ag assembly and regulation will be considered, such as locating a site on T-ag whose interactions with the flanking sequences in the core origin is necessary for T-ag binding. Collectively, these studies will provide information that may allow us to solve the mechanism of DNA unwinding. Once the SV40 origin is unwound, the pol alpha-primase complex initiates the synthesis of nascent DNA strands. Exactly what sequences constitute the initiation sites for the pol alpha-primase complex is the topic of an additional series of experiments. Given that many basic processes are conserved throughout evolution, we are confident that the information we obtain from these studies will be pertinent to the initiation of DNA replication at other viral origins of replication. Furthermore, they may help us to understand how the cell cycle machinery controls initiation events at cellular origins. Given that uncontrolled DNA replication is thought to be a component of many diseases, including cancer, it is very important to understand how DNA replication is initiated and how it is controlled.