Adeno-associated virus (AAV) site-specific integration: Formation of AAV-AAVS1 junctions in an in vitro system Journal Article
| Authors: | Dyall, J.; Szabo, P.; Berns, K. I. |
| Article Title: | Adeno-associated virus (AAV) site-specific integration: Formation of AAV-AAVS1 junctions in an in vitro system |
| Abstract: | An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His- Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo. |
| Keywords: | human tissue; gene sequence; human cell; polymerase chain reaction; chromosome 19; gene amplification; hela cell; hela cells; molecular cloning; cloning, molecular; dna; recombination, genetic; base sequence; binding site; helicase; dna primers; adenoviridae; adenovirus; nuclear factor; virus recombination; virus integration; genes, viral; adeno-associated virus; dependovirus; humans; human; priority journal; article; denaturation |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 96 |
| Issue: | 22 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1999-10-26 |
| Start Page: | 12849 |
| End Page: | 12854 |
| Language: | English |
| DOI: | 10.1073/pnas.96.22.12849 |
| PUBMED: | 10536011 |
| PROVIDER: | scopus |
| PMCID: | PMC23128 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 16 August 2016 -- Source: Scopus |
