Genetics of HSV DNA Replication

描述(申请人提供)：单纯疱疹病毒(HSV)是一种普遍存在的人类病原体，可导致严重的人类发病率和死亡率。包括病毒在内的所有有机体的生存取决于它们产生遗传物质的精确副本的能力。DNA复制机制(复制体)的功能是蛋白质的复杂结合，必须受到严格的调控，才能完成忠实和完整的DNA复制。我们之前的工作已经鉴定和鉴定了七种HSV复制蛋白以及细胞中发生DNA复制的隔室；然而，它们在DNA复制过程中发挥作用的机制尚不清楚。我们和我们的合作者最近的实验表明，HSV-1复制体的组装和功能受到蛋白质-蛋白质和蛋白质-核酸相互作用的严格调控，我们现在才开始意识到这一点。我们的中心假设是，病毒复制蛋白之间的蛋白质-蛋白质相互作用对于基因组复制的所有阶段都是必不可少的。我们将研究特定的相互作用在复制前位点和复制间隔的形成、病毒复制起始处DNA合成的启动、复制叉前的双链DNA的过程解开以及领先和滞后链DNA合成的协调调节中的作用。HSV戏剧性地重组感染的细胞核，导致形成大的球状复制间隔，在其中发生基因表达、DNA复制和囊化。虽然人们早就认识到ICP8是必要的，但人们对ICP8如何对核架构产生影响知之甚少。在……里面 目的1我们将结合遗传学、生物化学、生物物理学和细胞生物学的方法来验证ICP8的动态特性及其组装成小的亚组分和细丝的能力驱动复制前位点和复制室的形成的假说。已知ICP8和起源结合蛋白UL9在DNA合成的启动过程中共同作用来解开起源；然而，在体外重建依赖于起源的DNA合成的困难阻碍了对这一过程的深入了解。在目标2中，我们将检验这一假设，即蛋白质-蛋白质和蛋白质-DNA相互作用对于依赖来源的病毒DNA合成的启动是必不可少的。我们将使用遗传和生化方法来确定UL9和ICP8的区域，这些区域对起源解开的各个步骤都很重要。在目标3中，我们使用一种新开发的协调领先和滞后链合成的方法来检验这一假设，即HSV解旋酶/启动酶和聚合酶之间的相互作用是协调领先和滞后链DNA合成所必需的。

DESCRIPTION (provided by applicant): Herpes Simplex Virus (HSV) is a ubiquitous human pathogen that can cause significant morbidity and mortality in humans. The survival of all organisms including viruses depends on their ability to produce an exact copy of their genetic material. The DNA replication machinery (replisome) functions as a complex association of proteins that must be tightly regulated in order to accomplish faithful and complete DNA replication. Our previous work has led to the identification and characterization of seven HSV replication proteins as well as the compartments in the cell in which DNA replication occurs; however, the mechanisms by which they function during DNA replication are poorly understood. Recent experiments by us and our collaborators suggest that the assembly and function of the HSV-1 replisome is tightly regulated by protein-protein and protein-nucleic acid interactions that we are only now beginning to appreciate. Our central hypothesis is that protein-protein interactions among viral replication proteins are essential for all stages of genome replication. We will examine the roles of specific interactions in the formation of prereplicative sites and replication compartments, initiation of DNA synthesis at viral origins of replication, processive unwinding of the duplex DNA in front of the replication fork and the coordinated regulation of leading and lagging strand DNA synthesis. HSV dramatically reorganizes the infected cell nucleus leading to the formation of large globular replication compartments in which gene expression, DNA replication and encapsidation occur. Although it has long been recognized that ICP8 is required, little is known about how ICP8 exerts its effects on the nuclear architecture. In aim 1 we will use a combination of genetic, biochemical, biophysical and cell biological methods to test the hypothesis that dynamic properties of ICP8 and its ability to assemble into small subassemblies and filaments drive the formation of prereplicative sites and replication compartments. ICP8 and the origin binding protein UL9 are known to work together to unwind the origins during the initiation of DNA synthesis; however, difficulties in reconstituting origin dependent DNA synthesis in vitro have prevented a thorough understanding of the process. In aim 2 we will test the hypothesis that protein-protein and protein-DNA Interactions are essential for origin dependent initiation of viral DNA synthesis. We will use genetic and biochemical approaches to identify the regions of UL9 and ICP8 that are important for various steps in origin unwinding. In aim 3 we use a newly developed assay for coordinated leading and lagging strand synthesis to test the hypothesis that interactions between the HSV helicase/primase and polymerase are required for coordinated leading and lagging strand DNA synthesis.