ArticleJournal of virology1999
Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein.
Article in Journal of virology, 1999. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 40 citations in OpenAlex.
- Inducible HEK293 AAV packaging cell lines expressing Rep proteins.Molecular therapy. Methods & clinical development · 2023Article
- Applications of Deep Mutational Scanning in Virology.Viruses · 2021Review
- Article
- Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication.PloS one · 2017Article
- Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.Journal of virology · 2016Article
- Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding.Virology · 2015Article
- Rapid establishment of a HEK 293 cell line expressing FVIII-BDD using AAV site-specific integration plasmids.BMC research notes · 2014Article
- Mutations in DNA binding and transactivation domains affect the dynamics of parvovirus NS1 protein.Journal of virology · 2013Article
- The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization.Journal of virology · 2012Article
- The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage.International journal of biological sciences · 2012Article
- DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.PLoS pathogens · 2009Article
- Characterization of adeno-associated virus genomes isolated from human tissues.Journal of virology · 2005Article
- Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.Journal of virology · 2005Article
- Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey.Journal of virology · 2003Article
- Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat.Proceedings of the National Academy of Sciences of the United States of America · 2001Article
- Targeted integration of foreign DNA into a defined locus on chromosome 19 in K562 cells using AAV-derived components.International journal of hematology · 2001Article
- Amino-terminal domain exchange redirects origin-specific interactions of adeno-associated virus rep78 in vitro.Journal of virology · 2001Article
- Selective cleavage of AAVS1 substrates by the adeno-associated virus type 2 rep68 protein is dependent on topological and sequence constraints.Journal of virology · 2000Article
- An adeno-associated virus (AAV) initiator protein, Rep78, catalyzes the cleavage and ligation of single-stranded AAV ori DNA.Journal of virology · 2000Article
- A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay.Journal of virology · 2000Article
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Authors and funding
7 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.
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