AAV REP PROTEINS AND TARGETED INTEGRATION

AAV REP 蛋白质和靶向整合

• 批准号: 3757654
• 负责人: R KOTIN
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757654
• 关键词: DNA binding protein; Escherichia coli; adeno associated virus group; chromosomes; human genetic material tag; nucleic acid structure; protein structure function; provirus; virus protein; virus replication

Targeted integration of adeno-associated viral (AAV) DNA into human chromosome 19 occurs by nonhomologous recombination. The presence of the nonstructural protein (Rep) genes of AAV correlated with the specificity of proviral integration. Therefore, we investigated the involvement of Rep proteins in targeted integration. To facilitate the analysis of Rep protein functions, biologically active, recombinant Rep proteins were produced in E. coli. Binding of Rep to linear duplex DNA is a newly identified property of Rep which redefines the binding requirements for Rep proteins. The AAV DNA chromosome 19 integration locus is a specific substrate for Rep binding, establishing that Rep may act as an agent for targeted integration. The results of in vitro replication experiments demonstrated that a chromosome 19 subfragment may act as a Rep specific origin of replication. Thus we believe that in AAV infected cells, Rep promotes limited replication from the integration locus. The affinity of Rep proteins for either the viral or host substrate may permit Rep subunit exchange with the polymerase complex resulting in intermolecular DNA recombination.

将腺相关病毒 (AAV) DNA 靶向整合到人体中 19 号染色体通过非同源重组发生。 的存在 AAV 的非结构蛋白 (Rep) 基因与特异性相关 前病毒整合。 因此，我们调查了 靶向整合中的 Rep 蛋白。 为了方便Rep的分析 蛋白质功能、生物活性、重组Rep蛋白 在大肠杆菌中产生。 Rep 与线性双链 DNA 的结合是一种新的 Rep 的已识别属性重新定义了约束要求 代表蛋白质。 AAV DNA 19 号染色体整合位点是一个特定的 Rep 结合的底物，确定 Rep 可以充当 有针对性的整合。 体外复制实验结果 证明 19 号染色体亚片段可能充当 Rep 特异性 复制起点。 因此我们相信在 AAV 感染的细胞中，Rep 促进整合位点的有限复制。 亲和力 病毒或宿主底物的 Rep 蛋白可能允许 Rep 与聚合酶复合物的亚基交换导致分子间 DNA重组。