TARGETING OF AAV REP MEDIATED SPECIFIC DNA INTEGRATION

该项目的主要重点将是针对DNA 腺相关病毒Rep蛋白介导的整合 （AAV）。 如前所述，AAV整合其基因组位点特异性地 人类19号染色体 使用基于游离体的分析，我们能够 为了证明存在于靶内的33个核苷酸的信号 序列，是必要的，足以介导这种高度特异性的 整合/重组事件。 DNA靶向信号包含两个 序列基序也存在于病毒起源中， 复制，并由病毒Rep蛋白结合。 的 假设是病毒Rep蛋白决定位点特异性 通过识别和结合这些原始信号进行整合 存在于19号染色体上。 该项目将测试的可行性， 重定向Rep介导的位点特异性DNA整合， 交换AAV Rep蛋白的DNA结合结构域。 为了 建立了一个模型系统，我们首先提出了交换DNA结合 AAV Rep 78的结构域与其来自鹅细小病毒（GPV）的对应物。 有趣的是，两种蛋白质之间的相似性超过90 在Rep的活性结构域内的百分比。DNA结合结构域共享 相似性低得多，这与GPV Rep 1 识别不同的DNA基序 以下生化 为了表征杂合蛋白，重组AAV将被 产生含有编码杂合蛋白质的REP基因。这 病毒将用于基于附加体的整合测定，以测试 改变的位点特异性DNA整合到序列基序中， 被GPV Rep 1蛋白识别。 第二个目标是重新定向 整合到人类基因组内除AAVS 1以外的位点。 一 基因筛选将用于鉴定Rep蛋白的突变体 能够以高亲和力结合确定的细胞靶序列。 一旦这些突变蛋白被纯化并进行生化表征， 附加型整合分析将用于测试整合到 新的靶序列。 提出的实验旨在 为靶向DNA整合的机制提供了进一步的见解 关于AAV Rep的功能，以及为我们提供一个工具， 以人类基因组中的特定位点为目标。

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.