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Author Notes:

To whom correspondence should be addressed. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: xcheng@emory.edu

S.H. performed protein expression and purification, some DNA binding experiments, and crystallographic work;

D.W. performed some DNA binding experiments; J.R.H. participated in X-ray data collection;

X.Z. participated in discussion throughout and assisted in organizing and designing the study;

S.H.S. provided Zta construct; R.M.B. performed data analysis and assisted in preparing the manuscript;

X.C. organized and designed the scope of the study.

All were involved in analyzing data and in preparing the manuscript.

We thank B. Baker of New England Biolabs for synthesizing the oligonucleotides.

The Department of Biochemistry of Emory University School of Medicine supported the use of SER-CAT beamlines at the Advanced Photon Source, Argonne National Laboratory;

Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science.

Conflict of interest statement. None declared.


Research Funding:

National Institutes of Health (NIH) [GM049245-23 to X.C.]; Georgia Research Alliance Eminent Scholar [to X.C.].

The open access publication charge for this paper has been waived by Oxford University Press – NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal.


  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology

Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta

Journal Title:

Nucleic Acids Research


Volume 45, Number 5


, Pages 2503-2515

Type of Work:

Article | Final Publisher PDF


T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cβ atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/).

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