This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Urabe, M.
Right arrow Articles by Ozawa, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Urabe, M.
Right arrow Articles by Ozawa, K.

 Previous Article  |  Next Article 

Journal of Virology, April 1999, p. 2682-2693, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Charged-to-Alanine Scanning Mutagenesis of the N-Terminal Half of Adeno-Associated Virus Type 2 Rep78 Protein

Masashi Urabe,1,2,* Yoko Hasumi,1,2 Akihiro Kume,1,2 Richard T. Surosky,3 Gary J. Kurtzman,3 Kiyotake Tobita,4 and Keiya Ozawa1,2,5

Division of Genetic Therapeutics, Center for Molecular Medicine,1 Department of Virology,4 and Department of Hematology,5 Jichi Medical School, Tochigi 329-0498, and CREST, Japan Science and Technology Corporation, Saitama 332-0012,2 Japan, and Avigen, Inc., Alameda, California 945023

Received 12 August 1998/Accepted 12 December 1998

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.

* Corresponding author. Mailing address: Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minami-kawachi, Tochigi 329-0498, Japan. Phone: 81-285-58-7402. Fax: 81-285-44-8675. E-mail: murabe{at}jichi.ac.jp.

Journal of Virology, April 1999, p. 2682-2693, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Schnepp, B. C., Jensen, R. L., Chen, C.-L., Johnson, P. R., Clark, K. R. (2005). Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues. J. Virol. 79: 14793-14803 [Abstract] [Full Text]  
  • Jang, M. Y., Yarborough, O. H. III, Conyers, G. B., McPhie, P., Owens, R. A. (2005). Stable Secondary Structure near the Nicking Site for Adeno-Associated Virus Type 2 Rep Proteins on Human Chromosome 19. J. Virol. 79: 3544-3556 [Abstract] [Full Text]  
  • Yoon-Robarts, M., Linden, R. M. (2003). Identification of Active Site Residues of the Adeno-associated Virus Type 2 Rep Endonuclease. J. Biol. Chem. 278: 4912-4918 [Abstract] [Full Text]  
  • Amiss, T. J., McCarty, D. M., Skulimowski, A., Samulski, R. J. (2003). Identification and Characterization of an Adeno-Associated Virus Integration Site in CV-1 Cells from the African Green Monkey. J. Virol. 77: 1904-1915 [Abstract] [Full Text]  
  • Young, S. M. Jr., Samulski, R. J. (2001). Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat. Proc. Natl. Acad. Sci. USA 10.1073/pnas.241508998v1 [Abstract] [Full Text]  
  • Yoon, M., Smith, D. H., Ward, P., Medrano, F. J., Aggarwal, A. K., Linden, R. M. (2001). Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro. J. Virol. 75: 3230-3239 [Abstract] [Full Text]  
  • Lamartina, S., Ciliberto, G., Toniatti, C. (2000). Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints. J. Virol. 74: 8831-8842 [Abstract] [Full Text]  
  • Smith, R. H., Kotin, R. M. (2000). An Adeno-Associated Virus (AAV) Initiator Protein, Rep78, Catalyzes the Cleavage and Ligation of Single-Stranded AAV ori DNA. J. Virol. 74: 3122-3129 [Abstract] [Full Text]  
  • Davis, M. D., Wu, J., Owens, R. A. (2000). Mutational Analysis of Adeno-Associated Virus Type 2 Rep68 Protein Endonuclease Activity on Partially Single-Stranded Substrates. J. Virol. 74: 2936-2942 [Abstract] [Full Text]  
  • Cathomen, T., Collete, D., Weitzman, M. D. (2000). A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay. J. Virol. 74: 2372-2382 [Abstract] [Full Text]  
  • Rinaudo, D., Lamartina, S., Roscilli, G., Ciliberto, G., Toniatti, C. (2000). Conditional Site-Specific Integration into Human Chromosome 19 by Using a Ligand-Dependent Chimeric Adeno-Associated Virus/Rep Protein. J. Virol. 74: 281-294 [Abstract] [Full Text]  
  • Brister, J. R., Muzyczka, N. (1999). Rep-Mediated Nicking of the Adeno-Associated Virus Origin Requires Two Biochemical Activities, DNA Helicase Activity and Transesterification. J. Virol. 73: 9325-9336 [Abstract] [Full Text]  
  • Gavin, D. K., Young, S. M. Jr., Xiao, W., Temple, B., Abernathy, C. R., Pereira, D. J., Muzyczka, N., Samulski, R. J. (1999). Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants. J. Virol. 73: 9433-9445 [Abstract] [Full Text]  
  • Wu, J., Davis, M. D., Owens, R. A. (1999). Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins. J. Virol. 73: 8235-8244 [Abstract] [Full Text]  
  • Young, S. M. Jr., Samulski, R. J. (2001). Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat. Proc. Natl. Acad. Sci. USA 98: 13525-13530 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»