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Hairpin-end conformation of adeno-associated virus genome determines interactions with DNA-repair pathways

Gene Therapy volume 20, pages 686–693 (2013)Cite this article

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Abstract

The palindromic terminal repeats (TRs) of adeno-associated virus (AAV) form DNA hairpins (HPs) are essential for replication and for priming the conversion of single-stranded virion DNA to double strand. In recombinant AAV (rAAV) gene-delivery vectors, they are targets for the DNA-repair pathways leading to circularization, concatemerization and, infrequently, chromosomal integration. We investigated the effect of the TR HP on recombination by comparing specific DNA substrates transfected into wild-type and DNA-repair-deficient cells. DNA molecules with the TR sequences constrained in the T-shaped HP conformation at one or both ends were subject to a loss of gene expression, which was partially relieved in ataxia telangiectasia mutated (ATM−/−) cells. The ATM-dependent effect was mediated by transcriptional silencing of a subset of HP-containing molecules in cis rather than a loss of DNA, and was dependent on the specific T-shaped structure of the HP and not the primary sequence. DNA molecules with simple U-shaped HP ends were unaffected by ATM-dependent silencing. The silenced molecules remained in a linear conformation, in contrast to the expressed molecules, which were circularized. In the absence of ATM activity, this subset remained linear but was actively expressed. DNA molecules with the TR sequence in the open duplex conformation, or without TR sequences, were unaffected by ATM mutation and were predominantly converted to circular forms. A separate HP-specific effect in normal cells resulted in a loss of DNA substrate in the nucleus and was ATM independent. These results suggest that the presence of the HP structure on rAAV vector genomes subjects them to specific, and sometimes unproductive, DNA-repair/recombination pathways.

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References

  1. Donsante A, Miller DG, Li Y, Vogler C, Brunt EM, Russell DW et al. AAV vector integration sites in mouse hepatocellular carcinoma. Science 2007; 317: 477.

    Article  CAS  Google Scholar 

  2. Donsante A, Vogler C, Muzyczka N, Crawford JM, Barker J, Flotte T et al. Observed incidence of tumorigenesis in long-term rodent studies of rAAV vectors. Gene Ther 2001; 8: 1343–1346.

    Article  CAS  Google Scholar 

  3. Nakai H, Montini E, Fuess S, Storm TA, Meuse L, Finegold M et al. Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice. Mol Ther 2003; 7: 101–111.

    Article  CAS  Google Scholar 

  4. Duan D, Yue Y, Engelhardt JF . Consequences of DNA-dependent protein kinase catalytic subunit deficiency on recombinant adeno-associated virus genome circularization and heterodimerization in muscle tissue. J Virol 2003; 77: 4751–4759.

    Article  CAS  Google Scholar 

  5. Nakai H, Storm TA, Fuess S, Kay MA . Pathways of removal of free DNA vector ends in normal and DNA-PKcs-deficient SCID mouse hepatocytes transduced with rAAV vectors. Hum Gene Ther 2003; 14: 871–881.

    Article  CAS  Google Scholar 

  6. Song S, Lu Y, Choi YK, Han Y, Tang Q, Zhao G et al. DNA-dependent PK inhibits adeno-associated virus DNA integration. Proc Natl Acad Sci USA 2004; 101: 2112–2116.

    Article  CAS  Google Scholar 

  7. Song S, Laipis PJ, Berns KI, Flotte TR . Effect of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle. Proc Natl Acad Sci USA 2001; 98: 4084–4088.

    Article  CAS  Google Scholar 

  8. Choi VW, McCarty DM, Samulski RJ . Host cell DNA repair pathways in adeno-associated viral genome processing. J Virol 2006; 80: 10346–10356.

    Article  CAS  Google Scholar 

  9. Shrivastav M, De Haro LP, Nickoloff JA . Regulation of DNA double-strand break repair pathway choice. Cell Res 2008; 18: 134–147.

    Article  CAS  Google Scholar 

  10. Cataldi MP, McCarty DM . Differential effects of DNA double-strand break repair pathways on single-strand and self-complementary adeno-associated virus vector genomes. J Virol 2010; 84: 8673–8682.

    Article  CAS  Google Scholar 

  11. McCarty DM . Self-complementary AAV vectors; advances and applications. Mol Ther 2008; 16: 1648–1656.

    Article  CAS  Google Scholar 

  12. McCarty DM, Monahan PE, Samulski RJ . Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther 2001; 8: 1248–1254.

    Article  CAS  Google Scholar 

  13. Roth DB, Porter TN, Wilson JH . Mechanisms of nonhomologous recombination in mammalian cells. Mol Cell Biol 1985; 5: 2599–2607.

    Article  CAS  Google Scholar 

  14. Snyder RO, Samulski RJ, Muzyczka N . In vitro resolution of covalently joined AAV chromosome ends. Cell 1990; 60: 105–133.

    Article  CAS  Google Scholar 

  15. Ni TH, Zhou X, McCarty DM, Zolotukhin I, Muzyczka N . In vitro replication of adeno-associated virus DNA. J Virol 1994; 68: 1128–1138.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Choi VW, Samulski RJ, McCarty DM . Effects of adeno-associated virus DNA hairpin structure on recombination. J Virol 2005; 79: 6801–6807.

    Article  CAS  Google Scholar 

  17. Akinc A, Thomas M, Klibanov AM, Langer R . Exploring polyethylenimine-mediated DNA transfection and the proton sponge hypothesis. J Gene Med 2005; 7: 657–663.

    Article  CAS  Google Scholar 

  18. Rudolph C, Lausier J, Naundorf S, Muller RH, Rosenecker J . In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers. J Gene Med 2000; 2: 269–278.

    Article  CAS  Google Scholar 

  19. Carson CT, Schwartz RA, Stracker TH, Lilley CE, Lee DV, Weitzman MD . The Mre11 complex is required for ATM activation and the G2/M checkpoint. EMBO J 2003; 22: 6610–6620.

    Article  CAS  Google Scholar 

  20. Solovjeva LV, Svetlova MP, Chagin VO, Tomilin NV . Inhibition of transcription at radiation-induced nuclear foci of phosphorylated histone H2AX in mammalian cells. Chromosome Res 2007; 15: 787–797.

    Article  CAS  Google Scholar 

  21. Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH, Pepperkok R et al. RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 2009; 136: 435–446.

    Article  CAS  Google Scholar 

  22. Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB et al. RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell 2007; 131: 901–914.

    Article  CAS  Google Scholar 

  23. Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD et al. Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science 2007; 318: 1637–1640.

    Article  CAS  Google Scholar 

  24. Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 2004; 431: 873–878.

    Article  CAS  Google Scholar 

  25. Okazaki T, PD-1 Honjo T . and PD-1 ligands: from discovery to clinical application. Int Immunol 2007; 19: 813–824.

    Article  CAS  Google Scholar 

  26. Amiss TJ, McCarty DM, Skulimowski A, Samulski RJ . Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey. J Virol 2003; 77: 1904–1915.

    Article  CAS  Google Scholar 

  27. Wang Y, Zhu W, Levy DE . Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR. Methods 2006; 39: 356–362.

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Kimberly Zaraspe for technical assistance with this research. This work was supported by NIH grant R01 AI070244 to DMM.

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Authors and Affiliations

  1. Molecular, Cellular and Developmental Biology Program, The Ohio State University, Columbus, OH, USA

    M P Cataldi

  2. Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, USA

    D M McCarty

  3. Department of Pediatrics, The Ohio State University College of Medicine, Columbus, OH, USA

    D M McCarty

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  1. M P Cataldi
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  2. D M McCarty
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Correspondence to D M McCarty.

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Cataldi, M., McCarty, D. Hairpin-end conformation of adeno-associated virus genome determines interactions with DNA-repair pathways. Gene Ther 20, 686–693 (2013). https://doi.org/10.1038/gt.2012.86

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