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Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B

Nature Structural & Molecular Biology volume 24, pages 131139 (2017) | Download Citation

Abstract

APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded DNA (ssDNA) has beneficial functions in immunity and detrimental effects in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Although numerous structures have been solved, mechanisms for global ssDNA recognition and local target-sequence selection remain unclear. Here we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1-Å and 1.7-Å resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits into a groove between flexible loops and makes direct hydrogen bonds with the protein, accounting for the strong 5′-TC preference. These findings explain both conserved and unique properties among APOBEC family members, and they provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

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Acknowledgements

We thank D. Largaespada and D. Yee for insightful comments, R. Moorthy for oligonucleotide sample preparations, and J. Stivers (Pharmacology and Molecular Sciences Department, Johns Hopkins University, Baltimore, Maryland, USA) for providing the human UNG2 expression construct and purification protocol. This work was supported by grants from the US National Institutes of Health (NIGMS R01-GM118000 to R.S.H. and H.A., NIGMS R35-GM118047 to H.A., NIGMS R01-GM110129 to D.A.H., NCI R21-CA206309 to R.S.H., and DP2-OD007237 and NIGMS P41-GM103426 to R.E.A.), the NSF (CHE060073N to R.E.A.), the Prospect Creek Foundation (R.S.H. and D.A.H.), and the University of Minnesota Masonic Cancer Center (Spore-Program-Project-Planning Seed Grant to R.S.H.). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the US National Institutes of Health (NIGMS P41-GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. R.S.H. is supported as the Margaret Harvey Schering Land Grant Chair for Cancer Research and as an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Ke Shi
    •  & Michael A Carpenter

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota, USA.

    • Ke Shi
    • , Michael A Carpenter
    • , Nadine M Shaban
    • , Kayo Kurahashi
    • , Daniel J Salamango
    • , Jennifer L McCann
    • , Gabriel J Starrett
    • , Justin V Duffy
    • , Reuben S Harris
    •  & Hideki Aihara
  2. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

    • Ke Shi
    • , Michael A Carpenter
    • , Nadine M Shaban
    • , Kayo Kurahashi
    • , Daniel J Salamango
    • , Jennifer L McCann
    • , Gabriel J Starrett
    • , Justin V Duffy
    • , Daniel A Harki
    • , Reuben S Harris
    •  & Hideki Aihara
  3. Institute for Molecular Virology, University of Minnesota, Minneapolis, Minnesota, USA.

    • Ke Shi
    • , Michael A Carpenter
    • , Nadine M Shaban
    • , Kayo Kurahashi
    • , Daniel J Salamango
    • , Jennifer L McCann
    • , Gabriel J Starrett
    • , Justin V Duffy
    • , Reuben S Harris
    •  & Hideki Aihara
  4. Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA.

    • Michael A Carpenter
    • , Nadine M Shaban
    • , Daniel J Salamango
    • , Jennifer L McCann
    • , Gabriel J Starrett
    •  & Reuben S Harris
  5. Howard Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota, USA.

    • Michael A Carpenter
    •  & Reuben S Harris
  6. Northeastern Collaborative Access Team, Cornell University, Advanced Photon Source, Lemont, Illinois, USA.

    • Surajit Banerjee
  7. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California, USA.

    • Özlem Demir
    •  & Rommie E Amaro
  8. Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, USA.

    • Daniel A Harki

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Contributions

R.S.H. and H.A. conceived and designed the studies. K.S., N.M.S., K.K., J.V.D., and H.A. purified proteins and established crystallization conditions. S.B. collected X-ray diffraction data. K.S. solved the crystal structures. M.A.C., D.J.S., J.L.M., and G.J.S. performed the deep-deamination studies. M.A.C. performed biochemical experiments. D.A.H. designed modified DNA substrates. O.D. and R.E.A. provided computational and structural insights. K.S., M.A.C., N.M.S., R.S.H., and H.A. drafted the manuscript, and all authors contributed to revisions and figure preparation.

Competing interests

R.S.H. and D.A.H. are cofounders, shareholders, and consultants of ApoGen Biotechnologies Inc. H.A. and R.E.A. are consultants for ApoGen Biotechnologies Inc. R.E.A. is a cofounder of Actavalon Inc. The other authors declare no competing financial interests.

Corresponding authors

Correspondence to Reuben S Harris or Hideki Aihara.

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    Supplementary Figures 1–7

  2. 2.

    Supplementary Data Set 1

    Raw gel images for data in Figures 3e and 5a,b

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DOI

https://doi.org/10.1038/nsmb.3344

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