Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA

Sreejana Ray; Desiree Tillo; Nima Assad; Aniekanabasi Ufot; Christopher Deppmann; Stewart R Durell; Aleksey Porollo; Charles Vinson

doi:10.1016/j.bbrc.2018.05.080

Replacing C189 in the bZIP domain of Zta with S, T, V, or A changes DNA binding specificity to four types of double-stranded DNA

Biochem Biophys Res Commun. 2018 Jul 2;501(4):905-912. doi: 10.1016/j.bbrc.2018.05.080. Epub 2018 May 18.

Authors

Sreejana Ray¹, Desiree Tillo¹, Nima Assad¹, Aniekanabasi Ufot¹, Christopher Deppmann², Stewart R Durell³, Aleksey Porollo⁴, Charles Vinson⁵

Affiliations

¹ Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
² Department of Biology, University of Virginia, Charlottesville, VA, 22903, USA; Department of Cell Biology, University of Virginia, Charlottesville, VA, 22903, USA; Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, 22903, USA.
³ Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
⁴ Center for Autoimmune Genomics and Etiology and Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, 45229, USA.
⁵ Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA. Electronic address: vinsonc@mail.nih.gov.

Abstract

Zta is a bZIP transcription factor (TF) in the Epstein-Barr virus that binds unmethylated and methylated DNA sequences. Substitution of cysteine 189 of Zta to serine (Zta(C189S)) results in a virus that is unable to execute the lytic cycle, which was attributed to a change in binding to methylated DNA sequences. To learn more about the role of this position in defining sequence-specific DNA binding, we mutated cysteine 189 to four other amino acids, producing Zta(C189S), Zta(C189T), Zta(C189A), and Zta(C189V) mutants. Zta and mutants were used in protein binding microarray (PBM) experiments to evaluate sequence-specific DNA binding to four types of double-stranded DNA (dsDNA): 1) with cytosine in both strands (DNA(C|C)), 2) with 5-methylcytosine (5mC) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) with both cytosines in all CG dinucleotides containing 5mC (DNA(5mCG)). Zta(C189S) and Zta(C189T) bound the TRE (AP-1) motif (TGA^G/_CTCA) more strongly than wild-type Zta, while binding to other sequences, including the C/EBP half site GCAA was reduced. Binding of Zta(C189S) and Zta(C189T) to DNA containing modified cytosines (DNA(5mC|C), DNA(5hmC|C), and DNA(5mCG)) was reduced compared to Zta. Zta(C189A) and Zta(C189V) had higher non-specific binding to all four types of DNA. Our data suggests that position C189 in Zta impacts sequence-specific binding to DNA containing modified and unmodified cytosine.

Keywords: Cytosine methylation; DNA binding; Transcription factor; Zta; bZIP.

Published by Elsevier Inc.

Publication types

Research Support, N.I.H., Intramural

MeSH terms

5-Methylcytosine / analogs & derivatives
5-Methylcytosine / metabolism
Amino Acid Substitution*
Base Sequence
Basic-Leucine Zipper Transcription Factors / chemistry*
DNA / metabolism*
DNA Methylation / genetics
Mutant Proteins / chemistry
Nucleotide Motifs / genetics
Polymorphism, Single Nucleotide / genetics
Protein Binding
Protein Domains
Structure-Activity Relationship
Trans-Activators / chemistry*
Trans-Activators / metabolism*

Substances

BZLF1 protein, Herpesvirus 4, Human
Basic-Leucine Zipper Transcription Factors
Mutant Proteins
Trans-Activators
5-hydroxymethylcytosine
5-Methylcytosine
DNA

Grants and funding

Z99 CA999999/Intramural NIH HHS/United States