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The recombination signals for adeno-associated virus site-specific integration.

Linden, R M, Winocour, E, Berns, K I·1996·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.93.15.7966
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Abstract

The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.

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FiG. 1 Putative recombinogenic portions of the 510-bp 5' end of AAVS1. (A) Alignment of putative recombinogenic elements present in the AAV terminal repeats and in AAVS1. (B) Portions of AAVS1 cloned into p220.2 for assessment of their ability to serve as targets for AAV integration. The AAVS1 nucleotide positions are indicated in parentheses. Vertical lines indicate the positions of the putative recombination signals M26/cAMP response element (nt 275-283), Sp1 recognition site (nt 354–370), TRŠ (nt 384–389), and RBS (nt 398–413). (C) The oligonucleotide used for generating mutations within the RBS in pMUT17 is shown in alignment with the corresponding AAVS1 sequence. Seven nucleotides within the RBS were exchanged. The recombinogenic activity of the above AAVS1 segments is given in Table 1.
FiG. 1 Putative recombinogenic portions of the 510-bp 5' end of AAVS1. (A) Alignment of putative recombinogenic elements present in the AAV terminal repeats and in AAVS1. (B) Portions of AAVS1 cloned into p220.2 for assessment of their ability to serve as targets for AAV integration. The AAVS1 nucleotide positions are indicated in parentheses. Vertical lines indicate the positions of the putative recombination signals M26/cAMP response element (nt 275-283), Sp1 recognition site (nt 354–370), TRŠ (nt 384–389), and RBS (nt 398–413). (C) The oligonucleotide used for generating mutations within the RBS in pMUT17 is shown in alignment with the corresponding AAVS1 sequence. Seven nucleotides within the RBS were exchanged. The recombinogenic activity of the above AAVS1 segments is given in Table 1.From the PDF
FIG. 3 A model for the AAV site-specific DNA integration. Parallel lines in the AAV molecules are indicating that the sizes of DNA structures in the figure are not drawn in their actual proportions. A thick grey line indicates the newly synthesized strand; (Fig. 3 Īegend continues on the opposite page)
FIG. 3 A model for the AAV site-specific DNA integration. Parallel lines in the AAV molecules are indicating that the sizes of DNA structures in the figure are not drawn in their actual proportions. A thick grey line indicates the newly synthesized strand; (Fig. 3 Īegend continues on the opposite page)From the PDF

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Research works printed but not linked: 9

  1. [3]Hoggan, M. D., Blacklow, N. R. & Rowe, W. P. (1966) Proc. Natl. Acad. Sci. USA 55, 1467–1471.
  2. [12]Hoggan, M. D., Thomas, G. F. & Johnson, F. B. (1972) Continuous "carriage" of Adenovirus-Associated Virus Genomes in Cell Cultures in the Ábsence of Helper Adenovirus. (North-Holland, Amsterdam), pp. 243–249.
  3. [15]Berns, K. I. (1996) in Parvoviridae: The Viruses and Their Replication, eds. Fields, B. N., Knipe, D. M. & Howley, P. M. (Lippincott-Raven, Philadelphia), Vol. 2, pp. 2173–2220.
  4. [17]Kotin, R. M., Siniscalco, M., Šamulski, R. J., Zhu, X. D., Hunter, L., Laughlin, C. A., McLaughlin, S., Muzyczka, N., Rocchi, M. & Berns, K. I. (1990) Proc. Natl. Acad. Sci. USA 87, 2211–2215.
  5. [22]Giraud, C., Winocour, E. & Berns, K. I. (1994) Proc. Natl. Acad. Sci. USA 91, 10039–10043.
  6. [24]Straus, S. E., Sebring, E. D. & Rose, J. A. (1976) Proc. Natl. Acad. Sci. USA 73, 742–746.
  7. [30]Weitzman, M. D., Kyostio, S. R., Kotin, R. M. & Owens, R. A. (1994) Proc. Natl. Acad. Sci. USA 91, 5808–5812.
  8. [36]Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M. & Danielsen, M. (1987) Proc. Natl. Acad. Sci. USÀ 84, 7413–7417.
  9. [39]Ward, P. J. & Berns, K. I. (1996) J. Virol., in press.

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Authors
Linden, R. M., Winocour, E., & Berns, K. I.
Affiliations
Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, NY 10021, USA.
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
1996-07-23
Volume
93
Issue
15
Pages
7966-7972
ISSN
  • 0027-8424
  • 1091-6490
DOI
10.1073/pnas.93.15.7966
Cited by
211
References
0
Language
en

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