Mechanisms of the AAV2 Rep motor protein

AAV2 Rep 运动蛋白的机制

• 批准号: 7033560
• 负责人: RALPH MICHAEL LINDEN
• 金额: $ 32.67万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033560
• 关键词: DNA replication; adeno associated virus group; adenosine diphosphate; adenosine triphosphate; colorimetry; crystallization; enzyme activity; enzyme mechanism; enzyme structure; helicase; isomer; open reading frames; protein binding; protein structure function; thermodynamics; virus DNA; virus genetics; virus integration; virus protein; virus replication

DESCRIPTION (provided by applicant): Adeno-associated virus type 2 is an apparently non-pathogenic human virus that can infect cells in a cell cycle-independent fashion. The characteristics of AAV have been exploited in extensive efforts that have shown AAV to be among the most promising therapeutic gene transfer vehicles. In addition to the productive life cycle of AAV (that requires the presence of a helper virus such as adenovirus or herpes viruses) AAV has evolved the unique ability to integrate its genome site-specifically into human chromosome 19 in order to establish a latent infection. Our long-term goal is to elucidate the molecular mechanisms underlying AAV2 replication, site-specific integration and packaging. The small genome of AAV contains two open reading frames (REP and CAP). The Rep proteins orchestrate all aspects of the viral life style including DNA replication, site-specific integration, rescue from the integrated state and virus packaging. The study of this protein has been limited to biochemical characterizations and the lack of structural information has prevented a comprehensive depiction of the mechanistic aspects of Rep-mediated processes. Recently, we have solved the structure of the core helicase domain of AAV2 Rep. As this motor domain is present in all four Rep proteins it is likely to play an essential role in all aspects of Rep function. In this application we propose a collaborative and multifaceted approach (combining structural, biophysical and biochemical studies) aimed at understanding the structural basis for the motor activity of Rep within the context of the different isomers. Specifically, we will investigate the structural determinants of the Rep40 ATP-dependent motor activity (Aim 1); we will complement and extend the structural information by biochemical studies designed to help develop a model for the mechanism of Rep's motor activity (Aim 2); and, using Rep68, we will study the utilization of the motor activity that, in this context, is directed at replicative DNA unwinding (Aim 3).

描述(申请人提供)：腺相关病毒2型是一种明显非致病性的人类病毒，可以以细胞周期无关的方式感染细胞。AAV的特性已经在广泛的努力中得到了利用，这表明AAV是最有前途的治疗基因转移载体之一。除了AAV的生产性生命周期(需要腺病毒或疱疹病毒等辅助病毒的存在)外，AAV还进化出独特的能力，将其基因组位置整合到人类19号染色体中，以确定潜在感染。我们的长期目标是阐明AAV2复制、位点特异性整合和包装的分子机制。AAV的小基因组含有两个开放阅读框(REP和CAP)。Rep蛋白协调病毒生活方式的方方面面，包括DNA复制、位点特异性整合、从整合状态中拯救和病毒包装。对该蛋白质的研究仅限于生化特征，结构信息的缺乏阻碍了对Rep介导过程的机制方面的全面描述。最近，我们已经解决了AAV2Rp核心解旋酶结构域的结构，因为这个运动域存在于所有四个Rep蛋白中，它可能在Rep功能的各个方面发挥重要作用。在本申请中，我们提出了一种协作和多方面的方法(结合结构、生物物理和生化研究)，旨在了解不同异构体背景下Rep运动活性的结构基础。具体地说，我们将研究Rep40依赖于ATP的运动活动的结构决定因素(目标1)；我们将通过旨在帮助开发Rep运动活动机制的模型的生化研究来补充和扩展结构信息(目标2)；并且，利用Rep68，我们将研究在此背景下，针对复制DNA解旋的运动活动的利用(目标3)。