Initiation of SV40 DNA Replication and Its Regulation

使用肿瘤病毒，我们正在定义在DNA复制起始期间发生的事件。DNA复制的起始是一个复杂的过程，已知其发生在许多阶段;这些阶段包括病毒编码的起始蛋白对病毒起点的识别，随后与起点的结构畸变偶联的寡聚化事件，起始物转变为3 '-> 5'解旋酶和募集细胞因子用于随后的起始步骤。近年来，对T-ag结构的研究进展，有助于我们更好地理解它在催化DNA复制起始中的作用。我们将我们的结构研究集中在T-ag的结构域上，该位点特异性地识别了起源。使用X-射线晶体学，我们确定该结构域形成"螺旋六聚体"，并解决了与含有两个五核苷酸的起始亚片段结合的T-ag-obd的共结构。这些结构大大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，并为起源特异性解旋提供了重要的见解。然而，现在需要更多的结构信息，以了解启动过程。进一步的实验导致了β-发夹1的鉴定;一种存在于由广谱DNA肿瘤病毒编码的起始物中的基序。初步研究表明，该基序是必需的起源识别，双链体DNA的解链和随后的解旋酶活性。因此，我们将确定"β-发夹"在病毒复制起始期间所起的确切作用。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中起着积极的作用，并且在这个过程中产生的ssDNA是后续组装事件所需要的。已知BRCT结构域募集参与细胞周期控制、DNA修复和重组的蛋白质。我们最近的分析表明，BRCT家族成员存在于解旋酶域的T-ag。使用BRCT领域的个人开发的技术和方法，我们将确认BRCT基序存在于T-ag中。这一观察结果有可能极大地推进我们对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肿瘤病毒中DNA复制的起始是高度保守的。因此，在一个或两个模型系统中对启动的彻底描述将对我们理解不同病原体的传播产生广泛的影响。这些研究也将作为一个范例时，考虑如何启动DNA复制在高等真核生物。

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.