Half-site DNA Research Articles

ERRγ-DBD undergoes dimerization and conformational rearrangement upon binding to the downstream site of the DR1 element

The estrogen-related receptor (ERR) family members are reported to bind DNA elements as either monomer or dimer. However, to date, only one solution NMR structure of ERRβ in complex with a half-site DNA element has been reported. To better understand the DNA regulation mechanism, we determined the crystal structure of ERRγ-DBD bound to a natural DR1 element in Pla2g12b promoter to 2.2 Å resolution. Combined with biochemical assays, we show that ERRγ acts as a dimer and the C-terminal extension region undergoes conformational rearrangement when binding to the downstream DR1 element. In addition, the T-box region on the dimerization interface exhibits unique main-chain conformation. Thus, our structure presents a novel dimer interface for NR binding on DR1 DNA and provides a molecular basis for understanding the homodimer organization of ERR on DR1 elements.

A study of DNA design dependency of segmented DNA-induced gold nanoparticle aggregation towards versatile bioassay development

DNA-decorated gold nanoparticles (AuNPs) are important sensing probes for designing versatile assays for detecting DNA hybridization, DNA binders and DNA associated biological processes. In this study, we report DNA design rules for a pair of segmented DNA-conjugated AuNPs that can undergo “transient” aggregation due to cooperative base-pairing force of sticky ends on DNA and salt screening. A wildtype estrogen receptor response element (ERE) was used as a model DNA which is segmented into two half-sites, each carrying a complementary sticky end and a half-site DNA, were conjugated onto AuNPs to form two sets of complementary DNA–AuNPs conjugates. A number of DNA design parameters are studied for their effects on the aggregation kinetics, namely DNA conformation (i.e., mixed structure of ssDNA and dsDNA), number of sticky-ends bases, spacer length, as well as the symmetrical and asymmetrical interplay between the complementary set of segmented DNA–AuNPs conjugates. Firstly, we found that dsDNA serves as a more effective spacer than ssDNA in preventing base coordination of the nucleotides to the AuNPs surface due to its rigidity that in turn helps to improve the accessibility of sticky-ends for faster aggregation. Secondly, base-pairing force in facilitating the salt-induced AuNPs aggregation is tunable by the number of sticky-ends, which is closely related to the length and structural composition of the DNA spacer. Thirdly, symmetrically spaced sticky-ends enable quicker non-crosslinking aggregation than the asymmetrical combination due to their closer initial interparticle distance. Based on the optimized DNA design, we appended the efficient aggregation that leads to transient formation of a full ERE sequence for detecting estrogen receptor β (ER β) by exploiting protein binding retarded particle aggregation. With a competition assay, binding affinity of ERβ to different DNA sequences can be easily screened using a single set of complementary segmented DNA–AuNPs probes. The DNA design dependency of the DNA–AuNPs aggregation studied herein has enabled potential applications for rapid and highly specific DNA-binding protein detection, which could be extended to detect a wide range of DNA binders and its related biological processes.

A new and unexpected domain–domain interaction in the AraC protein

An interaction between the dimerization domains and DNA binding domains of the dimeric AraC protein has previously been shown to facilitate repression of the Escherichia coli araBAD operon by AraC in the absence of arabinose. A new interaction between the domains of AraC in the presence of arabinose is reported here, the regulatory consequences of which are unknown. Evidence for the interaction is the following: the dissociation rate of arabinose-bound AraC from half-site DNA is considerably faster than that of free DNA binding domain, and the affinity of the dimerization domains for arabinose is increased when half-site DNA is bound. In addition, an increase in the fluorescence intensity of tryptophan residues located in the arabinose-bound dimerization domain is observed upon binding of half-site DNA to the DNA binding domains. Direct physical evidence of the new domain-domain interaction is demonstrated by chemical crosslinking and NMR experiments.

Regulation of the human bile acid UDP-glucuronosyltransferase 1A3 by the farnesoid X receptor and bile acids

Cholestasis is a serious complication of many liver diseases leading to increased serum bile acids (BA) and their conjugates. Chenodeoxycholic (CDCA) acid is a substrate of the human hepatic UDP-glucuronosyltransferase (UGT) 1A3. UGT1A3 may, therefore, be a BA-inducible gene relevant to BA regulation. BA and human bile were used to induce UGT1A3 in HepG2 cells. Genomic DNA was analyzed by PCR amplification and sequencing. Transcriptional regulation was studied by DNA mutagenesis, RT-PCR, luciferase reporter gene constructs and electrophoretic mobility shift assays (EMSA). CDCA differentially induced UGT1A3 but not UGT1A4 expression. Bile from ursodeoxycholic acid (UDCA)-treated and untreated patients differentially induced UGT1A3. A farnesoid X receptor (FXR) half-site DNA motif was identified in the UGT1A3 5' upstream region. The FXR inducer GW4064 activated UGT1A3 transcription, and electrophoretic mobility shift assays identified UGT1A3 as a FXR target gene. Transcriptional regulation of the human bile acid and xenobiotic UGT1A3 by its substrate CDCA and FXR is shown. CDCA glucuronidation can be controlled by feed back inhibition proceeding via the glucuronidation of CDCA. UDCA does not induce UGT1A3 transcription. Since UGT1A3 is significantly induced by xenobiotics this physiologically links xenobiotic and bile acid metabolism to cholestasis.

Half-Site DNA Sequence and Spacing Length Contributions to PrrA Binding to PrrA Site 2 of RSP3361 in Rhodobacter sphaeroides 2.4.1

The consensus DNA binding sequence for PrrA, a global regulator in Rhodobacter sphaeroides 2.4.1, is poorly defined. We have performed mutational analysis of PrrA site 2, of the RSP3361 gene, to which PrrA binds in vitro (J. M. Eraso and S. Kaplan, J. Bacteriol. 191:4341-4352, 2009), to further define the consensus sequence for DNA binding. Two half-sites of equal length, containing 6 nucleotides each, were required for PrrA binding to this DNA sequence. Systematic nucleotide substitutions in both inverted half-sites led to a decrease in binding affinity of phosphorylated PrrA in vitro, the level of which was dependent on the substitution. The reduced binding affinities were confirmed by competition experiments and led to proportional decreases in the expression of lacZ transcriptional fusions to the RSP3361 gene in vivo. The 5-nucleotide spacer region between the half-sites was found to be optimal for PrrA binding to the wild-type half-sites, as shown by decreased PrrA DNA binding affinities to synthetic DNA sequences without spacer regions or with spacer regions ranging from 1 to 10 nucleotides. The synthetic spacer region alleles also showed decreased gene expression in vivo when analyzed using lacZ transcriptional fusions. We have studied three additional DNA sequences to which PrrA binds in vitro. They are located in the regulatory regions of genes positively regulated by PrrA and contain spacer regions with 5 or 8 nucleotides. We demonstrate that PrrA can bind in vitro to DNA sequences with different lengths in the spacer regions between the half-sites.

Structural Basis for p53 Binding-induced DNA Bending

Specific p53 binding-induced DNA bending has important biological implications such as transcription activation. However, the detailed structures of the bent DNA and the p53-DNA complex are still unavailable, hampering our understanding of the mechanism for p53-induced DNA bending and its consequent biological significance. To gain insight into the p53 binding-induced DNA bending, we performed molecular dynamics simulations on DNA segments with the consensus sequence for p53-specific binding, half site DNA-p53 complexes, and full site DNA-p53 complexes. We show that each DNA-bound p53 core domain caused a local DNA conformational change within the quarter site; upon the binding of the p53 dimer, there was an apparent DNA bending at the center of the half site; when bound with two p53 dimers, the full site DNAs with two different sequences bent 20 and 35 degrees, respectively. These results are in agreement with experimental observations. Our simulations demonstrate that the two p53 dimers favored a staggered conformation in which they make favorable interactions at the interface. This dimer-dimer interface organization necessitated conformational changes in the DNA, leading to the bending at the center of the full site, which in turn is dependent on the DNA sequence. Overall, our results provide the detailed atomic model for the DNA-p53 tetramer complex and delineate the roles of DNA-p53, p53 dimer-dimer interactions, and DNA sequence in specific p53 binding-induced DNA conformational changes.

Charge neutralization and DNA bending by the Escherichia coli catabolite activator protein.

We are interested in the role of asymmetric phosphate neutralization in DNA bending induced by proteins. We describe an experimental estimate of the actual electrostatic contribution of asymmetric phosphate neutralization to the bending of DNA by the Escherichia coli catabolite activator protein (CAP), a prototypical DNA-bending protein. Following assignment of putative electrostatic interactions between CAP and DNA phosphates based on X-ray crystal structures, appropriate phosphates in the CAP half-site DNA were chemically neutralized by methylphosphonate substitution. DNA shape was then evaluated using a semi-synthetic DNA electrophoretic phasing assay. Our results confirm that the unmodified CAP DNA half-site sequence is intrinsically curved by 26 degrees in the direction enhanced in the complex with protein. In the absence of protein, neutralization of five appropriate phosphates increases DNA curvature to 32 degrees (approximately 23% increase), in the predicted direction. Shifting the placement of the neutralized phosphates changes the DNA shape, suggesting that sequence-directed DNA curvature can be modified by the asymmetry of phosphate neutralization. We suggest that asymmetric phosphate neutralization contributes favorably to DNA bending by CAP, but cannot account for the full DNA deformation.

The Effect of C-Terminal Helix Stabilization on Specific DNA Binding by Monomeric GCN4 PeptidesReceived in Oxford, UK, 31st December 2001.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25, Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25°C in an aqueous buffer containing 100 mM NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide–DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1 : 1 complex with a dissociation constant of 1.41 ± 0.22 µM. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 ± 0.09 µM for peptide br-C and 3.9 ± 0.5 µM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the α-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd.

The Effect of C‐Terminal Helix Stabilization on Specific DNA Binding by Monomeric GCN4 Peptides

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.

The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half-site DNA: a NMR study

The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half-site 5′-TGCGG-3′. Apparent μM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74±7 ms at 20°C with the following DNA sequence: 5′-C1G2T3G4C5G6G7A8T9C10-3′. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half-site is another example of the variability in contacts of the fungal DNA-binding proteins containing Zn 2Cys 6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.

A model for the LexA repressor DNA complex.

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein-DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two beta-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed.

Recognition of DNA by GAL4 in Solution: Use of a Monomeric Protein-DNA Complex for Study by NMR

The complex of a monomer of GAL4 with DNA has been investigated by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Previous X-ray analysis has revealed a structure in which a dimer of the N-terminal 65-residue fragment of GAL4 forms a complex, 27 kDa in molecular mass, with a 19 base pair full-binding-site DNA [Marmorstein, R., Carey, M., Ptashne, M., & Harrison, S. C. (1992) Nature 356, 408-414]. We have developed a smaller system, half in molecular mass, which is amenable for detailed analysis using NMR. Titration of a 10 base pair half-binding-site DNA with GAL4-(65) shows 1:1 binding, illustrating that one monomer of the protein binds in a specific manner to half-site DNA. The components of the protein-DNA complex are mainly in fast exchange on the NMR chemical shift time scale, with an equilibrium dissociation constant of 161 +/- 12 microM. With a basis of chemical shift data for free GAL4 protein and for the free half-site DNA, the fast exchange facilitates 1H resonance assignments in the complex since cross-peak positions can be examined at different protein:DNA ratios. Chemical shift changes in the DNA reveal the base pairs that are important for recognition by GAL4. Intermolecular NOE cross-peaks are also observed in spectra of the protein-DNA complex. Their identification places the C-terminal end of the first alpha-helix (residues 12-17) in a position such that the amino acids are able to read the DNA sequence in a manner entirely consistent with the X-ray structure of the related complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of topoisomerase II-mediated DNA cleavage in the 5'-region of the Drosophila hsp70 gene. Identification of a novel half-site DNA substrate for topoisomerase II cleavage

Previous in vivo studies have identified a prominent 4'-demethylepipodophyllotoxin-9-(4,6-O-thionylidine-beta-D-g lucopyranoside) (VM-26)-induced double-stranded topoisomerase II cleavage site at approximately +80 relative to the start of Drosophila hsp70 transcription (Kroeger, P. E., and Rowe, T. C. (1992) Biochemistry 31, 2492-2502). Topoisomerase II binding at this site correlated with the repression of hsp70 transcription suggesting that this protein-DNA interaction was important in the regulation of hsp70 gene expression. In this paper, we investigated the interaction of purified Drosophila topoisomerase II with a 271-base pair DNA fragment containing the +80 region of the hsp70 gene using the topoisomerase II-specific inhibitor VM-26. VM-26-induced topoisomerase II cleavage of the hsp70 DNA resulted in a major 4-base staggered double-stranded break at +84. In the absence of ATP the +84 site was the only significant VM-26-induced cleavage site. Addition of ATP to the reaction resulted in a stimulation of topoisomerase cleavage throughout the 271-base pair DNA fragment. Deletion analyses determined that approximately 15 to 25 bp of flanking sequence were required for efficient cleavage at most topoisomerase II sites within the hsp70 DNA. However, in the case of the +84 site, topoisomerase cleavage still occurred even when this site was split in half by the restriction enzyme PstI. Topoisomerase II cleavage of both "half-site" DNA molecules occurred at the correct positions on the 4-base single-stranded DNA overhangs generated by PstI. Cleavage was reversible indicating that topoisomerase II could reseal the single-stranded DNA break formed in each half-site substrate. Denaturation of the half-site molecules abolished topoisomerase II cleavage suggesting that cleavage required the duplex region adjacent to the single-stranded cleavage site. Identification of this unusual half-site substrate provides additional evidence that double-stranded cleavage of DNA by topoisomerase II occurs via two sequential single-stranded breaks.