Coordination of HSV Lagging Strand Synthesis

描述(申请人提供)：双链DNA在一条链(前导)上连续复制，但在另一条(滞后)链上不连续复制，形成称为Okazaki片段的短片段。因此，滞后的链合成是一个复杂得多的过程。未能正确合成和处理Okazaki片段会导致突变增加、基因组不稳定和复制失败。真核生物中滞后的链合成是如何协调的，目前还知之甚少。单纯疱疹病毒(HSV)是一种遗传上易驯化的简单生物体，它编码DNA合成所需的大部分酶，但不是全部。指导本申请中提出的研究的首要假设是，冈崎片段的合成和处理需要HSV编码的片段和细胞酶活性的协调。在第一个目标中，我们将研究HSV-1启动酶活性与其聚合酶活性的协调以及UL5/UL52/UL8复合体的解旋酶活性与启动酶活性的协调要求。一系列含有启动引物合成的高效位点的半叉形和叉形DNA底物将检查装载UL5/UL52/UL8的最佳方向，与转位和分叉解离以及聚合酶延长引物的方向相比。在第二个目的中，将确定在冈崎片段终止后，触发进行中的HSV聚合酶向新的引物循环的条件和因素。在第三个目标中，将确定在最有效的体外条件下发生的蛋白质：蛋白质和蛋白质：DNA相互作用，以启动、终止或处理冈崎片段。此外，我们将使用全球蛋白质组学方法，通过质谱学鉴定与复制DNA特别相关的细胞和病毒蛋白质。提出了一种温和而特异地分离在细胞中以依赖于HSV来源的方式扩增的质粒的新方法。更好地了解HSV滞后链合成所需的步骤可能会导致干扰病毒复制周期的新策略。这个简单的模型系统也为理解哺乳动物细胞中对基因组稳定性至关重要的滞后链DNA的形成和处理提供了一个独特的机会。

DESCRIPTION (provided by applicant): Duplex DNA is copied continuously on one strand (leading), but discontinuously on the other (lagging) strand to form short segments called Okazaki fragments. Thus, lagging strand synthesis is a much more complex process. Failure to properly synthesize and process Okazaki fragments can lead to increased mutations, genomic instability, and replication failure. How lagging strand synthesis is coordinated in eukaryotes is poorly understood. Herpes simplex virus (HSV) is a genetically tractable and simple organism that encodes most, but not all, of the enzymes required for DNA synthesis. The overarching hypothesis guiding the studies proposed in this application is that synthesis and processing of Okazaki fragments require the coordination of HSV-encoded and cellular enzymatic activities. In the first aim, we will examine the requirements for coordination of the HSV-1 primase activity with its polymerase activity and for coordination of the helicase activity with primase activities of UL5/UL52/UL8 complex. A series of half-forked and forked DNA substrates that contain high efficiency sites for initiation of primer synthesis will examine the optimum orientation for loading UL5/UL52/UL8 for priming compared to that for translocation and fork unwinding, and for elongation of primers by polymerase. In the second aim, the conditions and factors that trigger the cycling of the processive HSV polymerase to new primers following termination of Okazaki fragments will be determined. In the third aim, the protein:protein and protein:DNA interactions that occur under the most efficient in vitro conditions for initiation, termination, or processing of Okazaki fragments will be determined. In addition, we will use a global proteomics approach to identify by mass spectrometry, cellular and viral proteins that specifically associate with replicating DNA. A novel approach to gently and specifically isolate plasmids that amplify in cells in an HSV origin-dependent manner is proposed. A better understanding of the steps required for HSV lagging strand synthesis could lead to novel strategies to disrupt the virus replication cycle. The simple model system also provides a unique opportunity to understand the formation and processing of lagging strand DNA in mammalian cells that is so vital to genomic stability.