Sequence-specific binding of [N-MeCys3,N-MeCys7]TANDEM to TpA.
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ABSTRACT:
SUBMITTER: Lavesa M
PROVIDER: S-EPMC1132212 | biostudies-other | 1993 Jan
REPOSITORIES: biostudies-other
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Project description:We have examined the dissociation of [N-MeCys3,N-MeCys7]TANDEM, an AT-selective bifunctional intercalator, from TpA sites in mixed-sequence DNAs by a modification of the footprinting technique. Dissociation of complexes between the ligand and radiolabelled DNA fragments was initiated by adding a vast excess of unlabelled calf thymus DNA. Portions of this mixture were subjected to DNAse I footprinting at various times after adding the competitor DNA. Dissociation of the ligand from each site was seen by the time-dependent disappearance of the footprinting pattern. Within a natural DNA fragment (tyrT) the ligand dissociates from TTAT faster than from ATAT. We found that the stability of complexes with isolated TpA steps decreases in the order ATAT > TTAA > TATA. Dissociation from each of these sites is much faster than from longer regions of (AT)n. These results confirm the requirement for A and T base-pairs surrounding the TpA step and suggest that the interaction is strongest with regions of alternating AT, possibly as a result of its unusual structure. The ligand dissociates more slowly from the centre of (AT)n tracts than from the edges, suggesting that variations in dissociation rate arise from sequence-dependent variations in local DNA structure.
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Project description:DNA binding protein are generally thought to bind specific DNA sequences through selective interactions with DNA bases. However, it is now becoming more widely appreciated that DNA shape, which may not be specified by a unique base sequence, also contributes to site-specific binding. Here we elucidate how DNA sequence and shape confer site specificity on a genomic scale, and relate this to specificity imparted indirectly through occlusion of sequences by the in vivo environment. For simplicity, we focus on the set of General Regulatory Factors (GRFs) that do not rely on other factors for binding. They also serve a related function in organizing chromatin. Remarkably, we find that GRFs will not bind to their cognate motif if the DNA surrounding that sequence lacks a specific shape. While proper DNA sequence/shape properties tend to be restricted to promoter regions, weaker sites that are still binding-competent reside in gene bodies, but are prevented from binding by resident chromatin. Thus, site-specificity is achieved across a genome in vivo by the combined action of favorable DNA sequence and shape interactions, and occlusion by chromatin.
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Project description: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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Project description:This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein-DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are essential for correct gene regulation. In this respect, ProteDNA is distinctive since it has been designed to identify sequence-specific binding residues. In order to accommodate users with different application needs, ProteDNA has been designed to operate under two modes, namely, the high-precision mode and the balanced mode. According to the experiments reported in this article, under the high-precision mode, ProteDNA has been able to deliver precision of 82.3%, specificity of 99.3%, sensitivity of 49.8% and accuracy of 96.5%. Meanwhile, under the balanced mode, ProteDNA has been able to deliver precision of 60.8%, specificity of 97.6%, sensitivity of 60.7% and accuracy of 95.4%. ProteDNA is available at the following websites: http://protedna.csbb.ntu.edu.tw/, http://protedna.csie.ntu.edu.tw/, http://bio222.esoe.ntu.edu.tw/ProteDNA/.
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Project description:The methodology of DNAase I footprinting has been adapted to investigate the sequence-specific binding of two quinoxaline drugs to DNA fragments containing natural and modified bases. In order to help comprehend the molecular origin of selectivity in the bis-intercalation of triostin A and TANDEM at CpG and TpA sites respectively, we have specifically examined the effect of the 2-amino group of guanine on their sequence specificity by using DNA in which that group has been either removed from guanine, added to adenine or both. Previous studies suggested that the recognition of particular nucleotide sequences by these drugs might be dependent upon the placement of the purine 2-amino group, serving as a positive or a negative effector for triostin A and TANDEM respectively. However, the footprinting data reported here indicate that this is not entirely correct, since they show that the 2-amino group of guanine is important for the binding of triostin A to DNA but has absolutely no influence on the interaction of TANDEM with TpA steps. Apparently the binding of triostin A to CpG sites is primarily due to hydrogen bonding interaction between the cyclic peptide of the antibiotic and the 2-amino group of guanine residues, whereas the selective binding of TANDEM to TpA sites is not hydrogen-bond driven and probably originates mainly from steric and/or hydrophobic interactions, perhaps involving indirect recognition of a suitable minor groove structure.
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