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| Status |
Public on Jun 04, 2019 |
| Title |
Mutating Zta (182N) changes sequence specific DNA binding (5mCG) |
| Platform organism |
synthetic construct |
| Sample organism |
human gammaherpesvirus 4 |
| Experiment type |
Other
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| Summary |
The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA. To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids: serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V). We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)). With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif. Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A. Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined. Zta(N182T) DNA binding is weaker to all types of DNA examined. Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).
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| Overall design |
Protein binding microarray (PBM) experiments were performed for the DNA binding domain of the Epstein Barr Virus Zta protein and for 5 mutant constructs of Zta in which the asparagine (N) at position 182 of wild type Zta was changed to serine (N182S), glutamine (N182Q), threonine (N182T), isoleucine (N182I), or valine (N182V). Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded and methylated or unmethylated 44K Agilent microarrays, containing a DeBruijn sequence design, in order to determine their sequence preferences. Details of the methylated PBM protocol are described in Mann et al., Genome Research, 2013
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| Contributor(s) |
Tillo D |
| Citation(s) |
35036684 |
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| Submission date |
Jun 05, 2018 |
| Last update date |
Jan 20, 2022 |
| Contact name |
Desiree Tillo |
| E-mail(s) |
desiree.tillo@nih.gov
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| Phone |
+1-240-760-7289
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| Organization name |
NIH/NCI
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| Street address |
41 Center Dr, Room D310
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (1) |
| GPL11260 |
Agilent custom ME and HK design array [8mer] |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA474675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE115352_RAW.tar |
24.2 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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