TARGETING OF AAV REP MEDIATED SPECIFIC DNA INTEGRATION

这个项目的主要焦点将是靶向DNA。 腺相关病毒Rep蛋白介导的整合 (AAV)。如前所述，AAV整合了其基因组位置--特别是 进入人类19号染色体。使用基于Episome的分析，我们能够 为了证明靶标中存在的33个核苷酸信号 序列，是调解这一高度特定的 整合/重组事件。DNA靶向信号包含两个 在病毒起源中也存在的序列基序 复制，并与病毒Rep蛋白结合。这个 假说是病毒Rep蛋白决定了位点特异性 通过识别和绑定这些起源信号进行整合 出现在19号染色体上。这个项目将测试 重定向Rep介导的位点特异性DNA整合 交换AAV Rep蛋白的DNA结合域。为了 建立模型系统，我们首先提出交换DNA结合 AAV Rep78的结构域与鹅细小病毒(GPV)的同源结构域。 有趣的是，这两种蛋白质之间的相似性超过90% 代表的活性结构域中的百分比。DNA结合结构域共享 更低的相似性与GPV Rep1的观察一致 识别出一个不同的DNA基序。接下来是生化 重组AAV杂合蛋白的鉴定将是 产生的含有编码杂交蛋白的rep基因。这 病毒将用于基于Episome的整合分析以测试 改变的位点特异性DNA整合到序列基序中，即 由GPV Rep1蛋白识别。第二个目标将是重新定向 整合到人类基因组中AAVS1以外的位置。一个 基因筛查将用于鉴定Rep蛋白的突变体 能够以高亲和力结合定义的细胞靶序列。 一旦这些突变蛋白被提纯并进行生化鉴定， 将使用附体整合试验来测试整合到 新的靶标序列。拟议中的实验旨在 为靶向DNA整合的机制提供进一步的见解 关于AAV代表的功能以及为我们提供的工具 以人类基因组中选定的位置为目标。

The primary focus of this project will be the targeting of DNA integration as mediated by the Rep proteins of adeno-associated virus (AAV). As shown previously, AAV integrates its genome site-specifically into human chromosome 19. Using an episome-based assay, we were able to demonstrate that a 33 nucleotide signal, present within the target sequence, is necessary and sufficient to mediate this highly specific integration/recombination event. The DNA targeting signal contains two sequence motifs which are also present in the viral origin of replication and which are bound by the viral Rep proteins. The hypothesis is that the viral Rep proteins determine site-specific integration through recognition and binding of these origin signals present on chromosome 19. This project will test the feasibility of retargeting Rep-mediated site-specific DNA integration by means of exchanging the DNA binding domain of the AAV Rep proteins. In order to establish a model system, we first propose to exchange the DNA binding domain of AAV Rep78 with its counterpart from goose parvovirus (GPV). Interestingly, the similarity between the two proteins exceeds 90 percent within the active domain of Rep. The DNA-binding domains share much lower similarity consistent with the observation that GPV Rep1 recognizes a different DNA motif. Following biochemical characterization of the hybrid protein a recombinant AAV will be generated containing a rep gene that encodes the hybrid protein. This virus will be used in an episome based integration assay to test for altered site-specific DNA integration into the sequence motif that is recognized by the GPV Rep1 protein. The second goal will be to redirect integration to sites within the human genome other than AAVS1. A genetic screen will be used to identify mutants of the Rep protein capable of binding defined cellular target sequences with high affinity. Once these mutant proteins are purified and biochemically characterized, the episomal integration assay will be used to test for integration into the new target sequences. The experiments proposed are designed to provide further insights into the mechanism of targeted DNA integration with regard to functions of AAV Rep as well as to provide us with a tool to target selected sites within the human genome.