Testing the Polyomavirus-based Replication Dependent Enhancer Duplication Model

当从健康个体中纯化时，大多数人多瘤病毒的调控区域含有单个增强子。然而，当从免疫受损个体的有效感染细胞中分离出来时，多瘤病毒的基因组含有促进病毒DNA复制和随后发病的复制增强子。然而，致病多瘤病毒中产生重复增强子的机制尚不清楚。为了解决这个问题，我们提出了一个多瘤病毒增强子复制的模型，该模型基于我们对以下方面的理解的进步；1)多瘤病毒DNA复制的开始，2)通过位移合成o - k - z - k - i - f - g - g - s形成“长瓣”，以及3)双链断裂修复所需的后续步骤。我们现在希望测试“复制依赖增强器复制”（RDED）模型的关键特性。关于将用于测试该模型的分析，先前的实验证实，在转染SV40病毒后，可以在CV1细胞中检测到增强子复制。因此，这是测试RDED模型的可行方法。然而，我们希望验证这样的假设，即增强子复制也会发生在更通用的基于质粒的复制分析中。一旦确定了最佳的细胞类型和检测方法（如PCR），我们将使用下一代测序（NGS）来定量增强子复制的频率，并确定在反应中形成的所有产物的序列。此外，我们将验证基于RDED的假设，即增强子复制将被冈崎片段形成所需因子的异常表达所促进。例如，一个含有强力霉素诱导的针对FEN-1的shRNA的细胞系将被用来检验含有新生DNA的“长瓣”将促进增强子复制的假设。在相关实验中，我们将验证阻断FEN1加载的发夹也会促进增强子复制的假设。额外的实验将测试基于red的假设，即在病毒起源附近的模板链上产生切口。此外，我们将验证核小体在限制增强子复制到调控区域中起主要作用的理论。最后，非病毒增强子也会受到复制和缺失的影响，这些改变会显著影响疾病基因的活性，包括致癌基因和肿瘤抑制基因。因此，我们对多瘤病毒中增强子复制形成的研究将对建立与多种人类疾病相关的增强子复制形成所需的机制具有重要作用。

When purified from healthy individuals, the regulatory regions of most human polyomaviruses contain single enhancers. However, when isolated from productively infected cells from immune- compromised individuals, the genomes of polyomaviruses contain duplicated enhancers that promote viral DNA replication and subsequent pathogenesis. The mechanism(s) that give rise to the duplicated enhancers in pathogenic polyomaviruses are, however, not known. To address this issue, we proposed a model for the duplication of the polyomavirus enhancers that is based upon advances in our understanding of; 1) the initiation of polyomavirus DNA replication, 2) the formation of “long flaps” via displacement synthesis o f O k a z a k i f r a g m e n t s and 3) the subsequent steps needed for double stranded break repair. We now wish to test key features of the “Replication Dependent Enhancer Duplication” (RDED) model. Regarding the assays that will be employed to test this model, previous experiments established that the enhancer duplications can be detected in CV1 cells following transfection of SV40 virus. Thus, this is a viable assay for testing the RDED model. However, we wish to test the hypothesis that the enhancer duplications will also occur in more versatile plasmid-based replication assays. Once the optimal cell type and assays (e.g., PCR) are established, we will use Next-generation sequencing (NGS) to quantitate the frequency of the enhancer duplications and to identify the sequences of all the products formed in the reactions. In addition, we will test the RDED- based hypothesis that the enhancer duplications will be promoted by the aberrant expression of factors needed for Okazaki fragment formation. For instance, a cell line containing a doxycycline inducible shRNA targeting FEN-1 will be used to test the hypothesis that the resulting “long flap” containing nascent DNA will promote the enhancer duplications. In related experiments, we will test the hypothesis that hairpins designed to block FEN1 loading will also promote the enhancer duplications. Additional experiments will test the RDED-based hypothesis that nicks are generated on the template strand in the vicinity of the viral origin. Moreover, we will test the theory that nucleosomes play a major role in limiting the enhancer duplications to the regulatory region. Finally, non-viral enhancers are also subjected to duplications and deletions and these alterations significantly impact the activity of disease genes, including oncogenes and tumor suppressors. Therefore, our studies of the formation of the enhancer duplications in polyomaviruses will have an important role in establishing the mechanism(s) needed to form the enhancer duplications associated with a wide variety of human diseases.