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THAP proteins target specific DNA sites through bipartite recognition of adjacent major and minor grooves

Nature Structural & Molecular Biology volume 17, pages 117–123 (2010)Cite this article

Abstract

THAP-family C2CH zinc-coordinating DNA-binding proteins function in diverse eukaryotic cellular processes, such as transposition, transcriptional repression, stem-cell pluripotency, angiogenesis and neurological function. To determine the molecular basis for sequence-specific DNA recognition by THAP proteins, we solved the crystal structure of the Drosophila melanogaster P element transposase THAP domain (DmTHAP) in complex with a natural 10-base-pair site. In contrast to C2H2 zinc fingers, DmTHAP docks a conserved β-sheet into the major groove and a basic C-terminal loop into the adjacent minor groove. We confirmed specific protein-DNA interactions by mutagenesis and DNA-binding assays. Sequence analysis of natural and in vitro–selected binding sites suggests that several THAPs (DmTHAP and human THAP1 and THAP9) recognize a bipartite TXXGGGX(A/T) consensus motif; homology suggests THAP proteins bind DNA through a bipartite interaction. These findings reveal the conserved mechanisms by which THAP-family proteins engage specific chromosomal target elements.

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Figure 1: Structure of DmTHAP–DNA complex and specific interactions with DNA.
Figure 2: Base-specific DmTHAP–DNA contacts.
Figure 3: Affinity determination of DmTHAP specificity mutants by EMSA.
Figure 4: Bipartite sequence readout by THAP proteins.
Figure 5: DmTHAP binds DNA in a manner distinct from the canonical zinc fingers.

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Acknowledgements

The authors would like to thank J. Gureasko and J. Kuriyan (Univ. California, Berkeley) for use of equipment and technical expertise, E. Abbate and M. Botchan (Univ. California, Berkeley) for crystallography supplies and experimental design, N. Echols and T. Alber (Univ. California, Berkeley) for use of equipment and assistance with data collection, J. Holton for assistance with data collection, A. May (Fluidigm) for crystallography supplies and assistance with data collection, D. King (Univ. California, Berkeley, and Howard Hughes Medical Institute Mass Spectrometry Laboratory) for MS analysis, N. Ogowa and M. Biggin (Univ. California, Berkeley) for reagents and expertise for the SELEX protocol, R. Schultzaberger and M. Eisen for assistance with SELEX data analysis and for creating the sequence logos, K. Collins for data analysis and D. Wemmer, M. Levine and M. Botchan for critical reading of the manuscript. A.Y.L. is supported by an American Cancer Society postdoctoral fellowship, J.M.B. by the US National Cancer Institute (CA077307) and D.C.R. by the National Institute of General Medical Sciences (GM61987).

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Author notes

  1. Alex Sabogal and Artem Y Lyubimov: These authors contributed equally to this work.

  2. jmberger@berkeley.edu

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Alex Sabogal, Artem Y Lyubimov, Jacob E Corn, James M Berger & Donald C Rio

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

    Artem Y Lyubimov, James M Berger & Donald C Rio

  3. Center for Integrative Genomics, University of California, Berkeley, Berkeley, California, USA

    Donald C Rio

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  1. Alex Sabogal
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Contributions

A.S. and D.C.R. conceived the experiments; D.C.R. synthesized brominated DNA oligonucleotides; A.S. purified the proteins and nucleic acids and crystallized the complex; J.E.C., A.Y.L. and J.M.B. provided guidance in crystallography trials; A.Y.L. and A.S. collected and analyzed the structural data; J.M.B. assisted with model building and refinement; A.Y.L. solved the structure and made all structural models; A.S. performed the DmTHAP mutagenesis and biochemistry, the SELEX experiment for human THAP9 and the THAP DNA-binding-site sequence analysis; A.S., A.Y.L., J.M.B. and D.C.R. wrote the paper.

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Correspondence to Donald C Rio.

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Sabogal, A., Lyubimov, A., Corn, J. et al. THAP proteins target specific DNA sites through bipartite recognition of adjacent major and minor grooves. Nat Struct Mol Biol 17, 117–123 (2010). https://doi.org/10.1038/nsmb.1742

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