Initiation of SV40 DNA Replication and Its Regulation

利用一种肿瘤病毒，我们正在定义DNA复制开始时发生的事件。DNA复制的起始是一个复杂的过程，已知发生在许多阶段；这包括病毒编码的启动蛋白对病毒起源的识别，随后与起源结构扭曲相结合的寡聚化事件，将启动物转变为3‘-> 5’解旋酶，以及为启动的后续步骤募集细胞因子。最近在确定T-ag结构方面的进展有助于我们了解其在催化DNA复制起始中的作用。我们的结构研究集中在T-ag的结构域上，该区域特异性识别了起源。利用x射线晶体学，我们确定该结构域形成了一个“螺旋六聚体”，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。这些结构极大地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，并为起源特异性解绕提供了关键的见解。然而，现在需要额外的结构信息来了解起始过程。进一步的实验鉴定出“β -发夹”；存在于由广泛的DNA肿瘤病毒编码的启动子中的基序。初步研究表明，这一基序对于DNA的起源识别、双链DNA的熔化以及随后的解旋酶活性都是必需的。因此，我们将确定“β -发夹”在病毒复制起始过程中所起的确切作用。例如，我们将扩展最近的研究，表明“β发夹”的尖端在熔化起源DNA中起积极作用，并且在此过程中产生的ssDNA是后续组装事件所需要的。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，一个BRCT家族成员存在于T-ag的解旋酶结构域。利用BRCT领域个人开发的技术和方法，我们将确认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。与公共卫生的相关性：最近的研究表明，DNA肿瘤病毒的DNA复制起始是高度保守的。因此，在一个或两个模型系统中对起始的彻底描述将对我们对各种病原体传播的理解产生广泛的影响。这些研究也将作为考虑DNA复制如何在高等真核生物中启动的范例。

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.