权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

AAV Rep-DNA Complexes Underlaying Site-specific Integration

AAV Rep-DNA 复合物是位点特异性整合的基础

基本信息

批准号：
8527803
负责人：
Carlos R Escalante
金额：
$ 27.85万
依托单位：
VIRGINIA COMMONWEALTH UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8527803
关键词：
Animal Viruses Architecture Binding Binding Sites Biochemical Cell Cycle Cells Chromosomes Chromosomes, Human, Pair 19 Complex DNA DNA biosynthesis DNA-Binding Proteins Dependovirus Development Elements Event Gene Transduction Agent Genetic Recombination Genome Goals Human Chromosomes Hydrolysis Kinetics Knowledge Lead Life Cycle Stages Mediating Modeling Nature Nonstructural Protein Pathogenicity Process Property Proteins Protocols documentation Reaction Recruitment Activity Regulation Replication Initiation Research Proposals Resolution Roentgen Rays Role Site Specificity Structure Traumeel S Virus Virus Replication X-Ray Crystallography base design gene therapy helicase insight latent infection melting new technology novel public health relevance research study site-specific integration stoichiometry structural biology tissue tropism tool viral DNA

项目摘要

DESCRIPTION (provided by applicant): Adeno-Associated Virus type 2 (AAV-2) is unique among animal viruses in its ability to establish a latent infection by integrating site-specifically into a locus of chromosome 19. Moreover, because of its apparent non- pathogenicity, broad cell and tissue tropism and ability to infect in a cell-cycle independent fashion, AAV has emerged as one of the most promising vectors for gene therapy. A number of studies indicate that AAV- mediated site-specific integration requires only three players: a) the AAV Rep78 or Rep68 non-structural proteins, b) an AAV DNA element containing a Rep Binding Site (RBS) and c) the cellular AAVS1 integration site. During a decisive event, these players come together to form a protein-DNA complex that serves as the starting point for an integration process that is considered to be parallel to the AAV replication initiation process. This pre-integration complex is dependent on the ability of Rep68/78 proteins to bind the integration site AAVS1, promote its melting and catalyze a strand-specific nicking reaction that leaves a Rep68/78 protein covalently bound to the integration site. Although a general view of this mechanism has been achieved through the study of AAV DNA replication, little is known regarding the architecture of the Rep-AAVS1 complex and the steps that lead to its formation. The long-term goal of this proposal is to establish a structural, biophysical and biochemical framework required to understand the mechanism of AAV-mediated site-specific integration. Our approach is to couple structural studies using X-ray crystallography, cryo-EM and small angle X-ray scattering (SAXS) with biophysical, kinetic and functional studies to get a complete understanding of the events that lead to site-specific integration. We expect that the results obtained in this research proposal will serve both as a starting point to generate accurate models to understand this unique mechanism and also will give us the knowledge and tools to potentially design Rep proteins with novel specificities that could be used to develop new technologies in the gene therapy field. PUBLIC HEALTH RELEVANCE: This study will contribute significantly toward understanding the mechanism of AAV mediated site-specific integration. In addition, it will contribute to our understanding of origin DNA replication initiation. Knowledge gained from this proposal will serve as the basis for designing novel gene therapy vectors that could be targeted to a specific region in the chromosome.

描述(申请人提供)：腺相关病毒2型(AAV-2)在动物病毒中是独一无二的，它通过将位置特异性整合到19号染色体上的一个位点来建立潜伏感染。此外，由于其明显的非致病性、广泛的细胞和组织趋向性以及以细胞周期无关的方式感染的能力，AAV已成为最有前途的基因治疗载体之一。许多研究表明，AAV介导的位点特异性整合只需要三个角色：a)AAVRep78或Rep68非结构蛋白，b)含有Rep结合位点(RBS)的AAVDNA元件，以及c)细胞AAVS1整合位点。在决定性的事件中，这些参与者聚集在一起形成蛋白质-DNA复合体，作为整合过程的起点，该过程被认为与AAV复制启动过程平行。这个整合前复合体依赖于Rep68/78蛋白与整合位点AAVS1结合的能力，促进其熔化，并催化链特异的缺口反应，使Rep68/78蛋白共价结合到整合位点。虽然通过对AAV DNA复制的研究已经对这一机制有了大致的了解，但对于Rep-AAVS1复合体的结构和导致其形成的步骤知之甚少。这项建议的长期目标是建立一个结构、生物物理和生化框架，以了解AAV介导的部位特异性整合的机制。我们的方法是将使用X射线结晶学、低温EM和小角X射线散射(SAXS)的结构研究与生物物理、动力学和功能研究结合起来，以全面了解导致特定部位整合的事件。我们希望在这项研究计划中获得的结果将作为一个起点，以产生准确的模型来理解这一独特的机制，并将为我们提供潜在的知识和工具，以设计具有新的特异性的Rep蛋白质，这些蛋白质可用于开发基因治疗领域的新技术。公共卫生相关性：这项研究将有助于理解AAV介导的部位特异性整合的机制。此外，它还将有助于我们对起源DNA复制起始的理解。从这项提议中获得的知识将作为设计新的基因治疗载体的基础，这些载体可以针对染色体上的特定区域。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.

腺相关病毒 Rep68 的寡聚特性反映了其多功能性。

DOI：
10.1128/jvi.02441-12
发表时间：
2013
期刊：
Journal of virology
影响因子：
5.4
作者：
Zarate-Perez,Francisco;Mansilla-Soto,Jorge;Bardelli,Martino;Burgner2nd,JohnW;Villamil-Jarauta,Maria;Kekilli,Demet;Samso,Monserrat;Linden,RMichael;Escalante,CarlosR
通讯作者：
Escalante,CarlosR

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy.

使用荧光各向异性测定腺相关病毒 Rep DNA 结合。

DOI：
10.21769/bioprotoc.2194
发表时间：
2017
期刊：
Bio-protocol
影响因子：
0.8
作者：
Zarate-Perez,Francisco;Santosh,Vishaka;Bardelli,Martino;Agundez,Leticia;Linden,RMichael;Henckaerts,Els;Escalante,CarlosR
通讯作者：
Escalante,CarlosR

The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

DOI：
10.1371/journal.ppat.1002764
发表时间：
2012
期刊：
PLoS pathogens
影响因子：
6.7
作者：
Zarate-Perez F;Bardelli M;Burgner JW 2nd;Villamil-Jarauta M;Das K;Kekilli D;Mansilla-Soto J;Linden RM;Escalante CR
通讯作者：
Escalante CR

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

DOI：
10.1021/acs.biochem.5b00610
发表时间：
2015-09-29
期刊：
Biochemistry
影响因子：
2.9
作者：
Musayev FN;Zarate-Perez F;Bardelli M;Bishop C;Saniev EF;Linden RM;Henckaerts E;Escalante CR
通讯作者：
Escalante CR

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.