Initiation of SV40 DNA Replication and Its Regulation

描述(申请人提供)：使用肿瘤病毒，我们正在定义在DNA复制启动过程中发生的事件。DNA复制的启动是一个复杂的过程，已知发生在许多阶段；这些包括病毒编码的启动子蛋白对病毒来源的识别，随后与起始点的结构扭曲相结合的寡聚事件，启动子向3‘->5’解旋酶的转变，以及为启动的后续步骤招募细胞因子。在确定T-Ag结构方面的最新进展有助于我们理解其在启动DNA复制中的作用。我们将结构研究的重点放在T-ag结构域上，该结构域专门识别起源。利用X射线结晶学，我们确定该结构域形成了一个‘螺旋-六角体’，并解决了T-Ag-OBD与含有两个五核苷酸的起始亚片段结合的共结构。这些结构显著增加了我们对病毒起源的结构安排、起源识别的精确机制的理解，并为特定起源的解开提供了关键的见解。然而，现在需要更多的结构信息来了解启动过程。其他实验导致了‘β-发夹1’的鉴定；这是一种存在于由广谱DNA肿瘤病毒编码的启动子中的基序。初步研究表明，这个基序是识别起源、融化双链DNA和随后的解旋酶活性所必需的。因此，我们将确定“β-发夹”在病毒复制启动过程中所扮演的确切角色(S)。例如，我们将扩大最近的研究，这些研究表明“β-发夹”的尖端在融化起源DNA方面起着积极的作用，并且在这个过程中产生的单链DNA是后续组装事件所必需的。已知BRCT结构域招募参与细胞周期控制、DNA修复和重组的蛋白质。我们最近的分析表明，在T-ag的解旋酶结构域中存在一个BRCT家族成员。使用BRCT领域中个人开发的技术和方法，我们将确认在T-ag中存在BRCT基序。这一观察结果有可能极大地促进我们对SV40如何导致其感染的细胞发生无数变化的理解。与公共卫生相关：最近的研究表明，DNA肿瘤病毒中DNA复制的启动是高度保守的。因此，彻底描述一个或两个模型系统中的启动将对我们理解不同病原体的传播产生广泛的影响。这些研究还将在考虑如何在高等真核生物中启动DNA复制时作为一个范例。

DESCRIPTION (provided by applicant): Using a tumor virus, we are defining the events that occur during the initiation of DNA replication. Initiation of DNA replication is a complicated process that is known to take place in many stages; these include the recognition of the viral origin by the virally encoded initiator protein, subsequent oligomerization events that are coupled to structural distortions of the origin, transition of the initiator into a 3'-> 5' helicase and recruitment of cellular factors for subsequent steps in initiation. Recent advances in the determination the structure of T-ag have contributed much to our understanding of its role in catalyzing the initiation of DNA replication. We have focused our structural studies on the domain of T-ag that site specifically recognized the origin. Using x-ray crystallography, we determined that this domain forms a 'spiral-hexamer' and solved the co-structure of the T-ag-obd bound to an origin sub-fragment containing two pentanucleotides. These structures have significantly increased our understanding of the architectural arrangement of viral origins, the precise mechanism of origin recognition and provided critical insights into origin specific unwinding. However, additional structural information is now needed in order to understand the initiation process. Additional experiments led to the identification of the 'beta-hairpin1; a motif that is present in the initiators encoded by a broad spectrum of DNA tumor viruses. Preliminary studies indicated that this motif is needed for origin recognition, melting of duplex DNA and subsequent helicase activities. Therefore, we will establish the exact role(s) played by the "beta-hairpin" during initiation of viral replication. For example, we will extend recent studies indicating that the tip of the "beta-hairpin" plays an active role in melting origin DNA and that the ssDNA generated in this process is needed for subsequent assembly events. BRCT domains are known to recruit proteins involved in cell-cycle control, DNA repair and recombination. Our recent analyses indicate that a BRCT family member is present in the helicase domain of T-ag. Using techniques and approaches developed by individuals in the BRCT field, we will confirm that a BRCT motif is present in T-ag. This observation has the potential to greatly advance our understanding of how SV40 causes the myriad changes in the cells that it infects. Relevance to Public Health: Recent studies have demonstrated that the initiation of DNA replication in DNA tumor viruses is highly conserved. Therefore, a thorough description of initiation in one or two model systems will have broad ramifications in terms of our understanding of the propagation of diverse pathogens. These studies will also serve as a paradigm when considering how DNA replication is initiated in higher-eukaryotic organisms.