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APOBEC3G DNA deaminase acts processively 3′ → 5′ on single-stranded DNA

Nature Structural & Molecular Biology volume 13, pages 392–399 (2006)Cite this article

Abstract

Akin to a 'Trojan horse,' APOBEC3G DNA deaminase is encapsulated by the HIV virion. APOBEC3G facilitates restriction of HIV-1 infection in T cells by deaminating cytosines in nascent minus-strand complementary DNA. Here, we investigate the biochemical basis for C → U targeting. We observe that APOBEC3G binds randomly to single-stranded DNA, then jumps and slides processively to deaminate target motifs. When confronting partially double-stranded DNA, to which APOBEC3G cannot bind, sliding is lost but jumping is retained. APOBEC3G shows catalytic orientational specificity such that deamination occurs predominantly 3′ → 5′ without requiring hydrolysis of a nucleotide cofactor. Our data suggest that the G → A mutational gradient generated in viral genomic DNA in vivo could result from an intrinsic processive directional attack by APOBEC3G on single-stranded cDNA.

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Figure 1: APOBEC3G purified from Sf9 cells is associated with RNA.
Figure 2: Effects of DNA length on APOBEC3G binding and catalysis.
Figure 3: Location of a CCC hot spot on an ssDNA substrate (69 nt) affects APOBEC3G activity.
Figure 4: APOBEC3G cycles on and off an ssDNA substrate.
Figure 5: Processive deamination of two C targets on the same ssDNA molecule.
Figure 6: Effect of a blocking dsDNA on APOBEC3G processivity.
Figure 7: Jumping and sliding model for APOBEC3G-catalyzed processive deamination.

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References

  1. Sheehy, A.M., Gaddis, N.C., Choi, J.D. & Malim, M.H. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646–650 (2002).

    Article  CAS  Google Scholar 

  2. Kobayashi, M., Takaori-Kondo, A., Miyauchi, Y., Iwai, K. & Uchiyama, T. Ubiquitination of APOBEC3G by an HIV-1 Vif-Cullin5-Elongin B-Elongin C complex is essential for Vif function. J. Biol. Chem. 280, 18573–18578 (2005).

    Article  CAS  Google Scholar 

  3. Conticello, S.G., Harris, R.S. & Neuberger, M.S. The Vif protein of HIV triggers degradation of the human antiretroviral DNA deaminase APOBEC3G. Curr. Biol. 13, 2009–2013 (2003).

    Article  CAS  Google Scholar 

  4. Beale, R.C. et al. Comparison of the differential context-dependence of DNA deamination by APOBEC enzymes: correlation with mutation spectra in vivo. J. Mol. Biol. 337, 585–596 (2004).

    Article  CAS  Google Scholar 

  5. Suspene, R. et al. APOBEC3G is a single-stranded DNA cytidine deaminase and functions independently of HIV reverse transcriptase. Nucleic Acids Res. 32, 2421–2429 (2004).

    Article  CAS  Google Scholar 

  6. Yu, Q. et al. Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV genome. Nat. Struct. Mol. Biol. 11, 435–442 (2004).

    Article  CAS  Google Scholar 

  7. Lecossier, D., Bouchonnet, F., Clavel, F. & Hance, A.J. Hypermutation of HIV-1 DNA in the absence of the Vif protein. Science 300, 1112 (2003).

    Article  CAS  Google Scholar 

  8. Harris, R.S. et al. DNA deamination mediates innate immunity to retroviral infection. Cell 113, 803–809 (2003).

    Article  CAS  Google Scholar 

  9. Mangeat, B. et al. Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts. Nature 424, 99–103 (2003).

    Article  CAS  Google Scholar 

  10. Zhang, H. et al. The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA. Nature 424, 94–98 (2003).

    Article  CAS  Google Scholar 

  11. Jarmuz, A. et al. An anthropoid-specific locus of orphan C to U RNA-editing enzymes on chromosome 22. Genomics 79, 285–296 (2002).

    Article  CAS  Google Scholar 

  12. Huthoff, H. & Malim, M.H. Cytidine deamination and resistance to retroviral infection: Towards a structural understanding of the APOBEC proteins. Virology 334, 147–153 (2005).

    Article  CAS  Google Scholar 

  13. Harris, R.S. & Liddament, M.T. Retroviral restriction by APOBEC proteins. Nat. Rev. Immunol. 4, 868–877 (2004).

    Article  CAS  Google Scholar 

  14. Xie, K. et al. The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1. Proc. Natl. Acad. Sci. USA 101, 8114–8119 (2004).

    Article  CAS  Google Scholar 

  15. Navarro, F. et al. Complementary function of the two catalytic domains of APOBEC3G. Virology 333, 374–386 (2005).

    Article  CAS  Google Scholar 

  16. Luo, K. et al. Amino-terminal region of the human immunodeficiency virus type 1 nucleocapsid is required for human APOBEC3G packaging. J. Virol. 78, 11841–11852 (2004).

    Article  CAS  Google Scholar 

  17. Hache, G., Liddament, M.T. & Harris, R.S. The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain. J. Biol. Chem. 280, 10920–10924 (2005).

    Article  CAS  Google Scholar 

  18. Shindo, K. et al. The enzymatic activity of CEM15/Apobec-3G is essential for the regulation of the infectivity of HIV-1 virion but not a sole determinant of its antiviral activity. J. Biol. Chem. 278, 44412–44416 (2003).

    Article  CAS  Google Scholar 

  19. Chiu, Y.L. et al. Cellular APOBEC3G restricts HIV-1 infection in resting CD4+ T cells. Nature 435, 108–114 (2005).

    Article  CAS  Google Scholar 

  20. Berg, O.G., Winter, R.B. & von Hippel, P.H. Diffusion-driven mechanisms of protein translocation on nucleic acids. 1. Models and theory. Biochemistry 20, 6929–6948 (1981).

    Article  CAS  Google Scholar 

  21. Halford, S.E. & Marko, J.F. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res. 32, 3040–3052 (2004).

    Article  CAS  Google Scholar 

  22. Stanford, N.P., Szczelkun, M.D., Marko, J.F. & Halford, S.E. One- and three-dimensional pathways for proteins to reach specific DNA sites. EMBO J. 19, 6546–6557 (2000).

    Article  CAS  Google Scholar 

  23. von Hippel, P.H. & Berg, O.G. Facilitated target location in biological systems. J. Biol. Chem. 264, 675–678 (1989).

    CAS  Google Scholar 

  24. Bransteitter, R., Pham, P., Scharff, M.D. & Goodman, M.F. Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100, 4102–4107 (2003).

    Article  CAS  Google Scholar 

  25. Creighton, S., Bloom, L.B. & Goodman, M.F. Gel fidelity assay measuring nucleotide misinsertion, exonucleolytic proofreading, and lesion bypass efficiencies. Methods Enzymol. 262, 232–256 (1995).

    Article  CAS  Google Scholar 

  26. Zhou, H.X. A model for the mediation of processivity of DNA-targeting proteins by nonspecific binding: dependence on DNA length and presence of obstacles. Biophys. J. 88, 1608–1615 (2005).

    Article  CAS  Google Scholar 

  27. Bransteitter, R., Pham, P., Calabrese, P. & Goodman, M.F. Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase. J. Biol. Chem. 279, 51612–51621 (2004).

    Article  CAS  Google Scholar 

  28. Pham, P., Bransteitter, R., Petruska, J. & Goodman, M.F. Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107 (2003).

    Article  CAS  Google Scholar 

  29. Bertram, J.G., Bloom, L.B., O'Donnell, M. & Goodman, M.F. Increased dNTP binding affinity reveals a nonprocessive role for Escherichia coli beta clamp with DNA polymerase IV. J. Biol. Chem. 279, 33047–33050 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful for the extensive expert advice provided by P. von Hippel and D. Astumian. We would like to thank R. Bransteitter for generating baculovirus expressing GST-APOBEC3G. This work was supported by US National Institutes of Health grants ESO13192 and R37GM21422.

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Authors and Affiliations

  1. Department of Biological Sciences Molecular and Computational Section, University of Southern California, Los Angeles, 90089-2910, California, USA

    Linda Chelico, Phuong Pham, Peter Calabrese & Myron F Goodman

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  1. Linda Chelico
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  2. Phuong Pham
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Correspondence to Myron F Goodman.

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Supplementary information

Supplementary Fig. 1

Deamination on substrates with 3 and 7 nt between two C targets (PDF 112 kb)

Supplementary Fig. 2

Processive deamination in the presence of competitor ssDNA (PDF 129 kb)

Supplementary Fig. 3

Processive deamination on 157-nt substrates (PDF 129 kb)

Supplementary Fig. 4

Processive deamination of hot spot and cold spots (PDF 143 kb)

Supplementary Table 1

List of synthetic oligonucleotides (PDF 138 kb)

Supplementary Methods

Synthesis of DNA substrates with internal γ-32P label (PDF 81 kb)

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Chelico, L., Pham, P., Calabrese, P. et al. APOBEC3G DNA deaminase acts processively 3′ → 5′ on single-stranded DNA. Nat Struct Mol Biol 13, 392–399 (2006). https://doi.org/10.1038/nsmb1086

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