![]() ![]() (18) We will first describe the results for each of the five methods to binding unmodified dsDNA and then binding to three dsDNAs containing modified cytosine. This allows us to determine the contribution to binding of all nucleotides, including modified cytosines, at each position in the motif. Fifth, we examined the effects of single nucleotide variants (SNVs) on binding the canonical TRE ( T –4G –3A -2G/ C 0T 2C 3A 4) motif 7-mer and related 7-mers containing one or no cytosines. Fourth, we generated dsDNA logos (18) using the 50 strongest bound 8-mers. This measure is sensitive to changes in median binding. The Z-score for a given 8-mer is the number of standard deviations of the intensity of that 8-mer (computed from all features containing the 8-mer) that is from the global median intensity (computed from all array features). Third, we determined a standardized score ( Z-score) (25) for binding 8 bp long dsDNA sequences (8-mers). Second, we examined the dependence on nucleotide and dinucleotide composition on binding. First, for an overall quantitative measure of binding, we examined the fluorescence intensities of the 40,330 features in each of the 16 sectors on the glass slide. (18) Binding was detected using a Cy5 conjugated antibody to the GST epitope followed by the measurement of fluorescence intensities at each of the array features. (24) Equal amounts of in vitro synthesized chimeric protein containing GST and bZIP domains ( Figure S2) were bound to these four types of dsDNA. (18,20,23) The fourth type of dsDNA was generated by enzymatic methylation of both cytosines in all CG dinucleotides ((DNA(5mCG)). T7 DNA polymerase was used to generate three types of dsDNAs: (i) dsDNA with cytosine on both strands (DNA(C|C)) and (ii, iii) dsDNA with 5mC or 5hmC in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)). (18) We examined the sequence-specific dsDNA binding of six GST-Zta chimeric constructs, Zta and five single amino acid mutants (Q, S, I, V, or T) of Zta(N182) to four types of dsDNA. Protein binding microarray (PBM) experiments used Agilent DNA microarrays containing 40,330 different single-stranded DNA 60-mers (22) in 16 sectors on a glass microarray slide. Molecular dynamics simulations rationalize some of these results, identifying hydrogen bonds between glutamine and A 3, but do not reveal why serine preferentially binds G 3, suggesting that entropic interactions may mediate this new binding specificity. ![]() Zta(N182V) and Zta(N182I) have increased binding to dsDNA(5mC|C). Zta(N182S) and Zta(N182Q) have 34- and 17-fold weaker median dsDNA binding, respectively. Zta(N182S) changes binding to G 3 in one or both halves of the motif. Zta (N182Q) changes binding to A 3 in only one half-site. Zta preferentially binds the pseudo-palindrome TRE (AP1) motif ( T –4G –3A -2G/ C 0T 2C 3A 4). We replaced asparagine with five similarly sized amino acids (glutamine (Q), serine (S), threonine (T), isoleucine (I), or valine (V)) and used protein binding microarrays to evaluate sequence-specific dsDNA binding. We studied the contribution of a conserved asparagine (N182) to sequence-specific dsDNA binding to four types of dsDNA: (i) dsDNA with cytosine in both strands ((DNA(C|C)), (ii, iii) dsDNA with 5-methylcytosine (5mC, M) or 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand ((DNA(5mC|C) and DNA(5hmC|C)), and (iv) dsDNA with methylated cytosine in both strands in all CG dinucleotides ((DNA(5mCG)). Zta, the Epstein–Barr virus bZIP transcription factor (TF), binds both unmethylated and methylated double-stranded DNA (dsDNA) in a sequence-specific manner. ![]()
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