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PubMed1995

Hydration patterns and intermolecular interactions in A-DNA crystal structures. Implications for DNA recognition.

Crystallographic studies of DNA fragments of the A and B conformations have shown that the structure and hydration of the DNA double helix depend both on the base sequence and the environment. Detailed analyses of solvent organization in DNA crystals and its role in intermolecular interactions have been reported mainly for B-DNA structures. We have determined the crystal structures of several isomorphous A-DNA octamers at resolutions from 1.8 to 2.5 A and refined them by the same procedure. Comparative analysis of five independently refined structures in terms of hydration and intermolecular interactions has been performed leading to the following findings. The A-DNA major groove is extensively hydrated and together with the hydration shells of the sugar-phosphate backbone can form an ordered network of fused polygons. The water structure of the phosphate backbone is less conserved than that of the grooves. Characteristic hydration patterns are associated with specific base sequences. The A-DNA minor groove provides sites for intermolecular contacts through hydrophobic and polar interactions. Well-ordered water molecules mediate interduplex interactions that involve either the grooves or the backbone, or both. The direct and water-mediated intermolecular interactions observed in the A-DNA crystal structures are relevant to various recognition motifs between DNA and other molecules. In particular, intermolecular interactions at the DNA minor groove are analogous to those observed in the recently reported crystal structures of complexes between the TATA-binding protein and the TATA-box.

Base Sequence↗
PubMed1993

Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization subdomains of novel structure.

EBNA-1 is essential for replication of the latent episomal form of the Epstein-Barr virus genome and is involved in regulation of viral latency promoters. EBNA-1 activity is mediated through direct DNA binding. The DNA binding and dimerization functions of EBNA-1 have previously been located to a carboxy-terminal domain, amino acids (aa) 459 to 607. To identify and define the subdomains for these two functions, we created an extensive series of deletions and point mutations in an EBNA-1 (aa 408 to 641) background. The ability of the EBNA-1 mutants to heterodimerize with a wild-type EBNA-1 (aa 459 to 641) Immunoprecipitation assays with a monoclonal antibody, EBNA.OT1x, that recognizes EBNA-1 (aa 408 to 641) but not EBNA-1 (aa 459 to 641). These experiments revealed that mutations affecting dimerization occurred over two separate regions, aa 501 to 532 and aa 554 to 598. DNA binding was tested in mobility shift assays against a panel of oligonucleotide-binding sites. Dimerization was a prerequisite for DNA binding. The DNA recognition domain was localized to a separate region, aa 459 to 487, upstream of the dimerization domain. EBNA-1 variants carrying substitutions at aa 467 and 468 and at aa 477 gave a pattern of binding to mutant oligonucleotide probes that implicates these particular amino acids in DNA recognition. EBNA-1 appears to utilize novel mechanisms for both DNA recognition and dimerization since neither domain conforms to previously described structural motifs.

Amino Acid Sequence↗
PubMed1994

Delineation of a 16 amino acid sequence that forms a core DNA recognition motif in the Epstein-Barr virus EBNA-1 protein.

EBNA-1 is the sole virally specified protein required for replication of the Epstein-Barr virus latency origin and also modulates its own expression. Both of these functions are mediated through specific DNA binding. We created a series of amino acid substitutions across the previously identified DNA-binding domain of EBNA-1 to further define the amino acids required for DNA recognition. The results of electrophoretic mobility shift assays using in vitro-translated EBNA-1 (aa 408-641) polypeptides indicated that: (i) The predicted alpha helical segment between aa 477 and 487 is not directly involved in DNA recognition but appears to contribute to a critical local polypeptide conformation. (ii) The positively charged residues Arg459, Lys460, and Lys461 are dispensable for DNA binding. (iii) The region between Gly462 and Lys477 contains residues important for DNA recognition. This region is extremely sensitive to mutation. The behavior of a synthetic peptide representing EBNA-1 aa 458-478 supported the mutagenesis data. A dimer form of this peptide, which lacks the predicted alpha helical domain, was capable of binding DNA, but only nonspecifically. The effect of binding affinity on transactivation was examined in cotransfection assays. EBNA-1 mutants with reduced binding affinity also demonstrated reduced levels of transactivation.

Amino Acid Sequence↗
PubMed2005

Integration of bioinformatics and computational biology to understand protein-DNA recognition mechanism.

Transcription factors play essential role in the gene regulation in higher organisms, binding to multiple target sequences and regulating multiple genes in a complex manner. In order to decipher the mechanism of gene regulation, it is important to understand the molecular mechanism of protein-DNA recognition. Here we describe a strategy to approach this problem, using various methods in bioinformatics and computational biology. We have used a knowledge-based approach, utilizing rapidly increasing structural data of protein-DNA complexes, to derive empirical potential functions for the specific interactions between bases and amino acids as well as for DNA conformation, from the statistical analyses on the structural data. Then these statistical potentials are used to quantify the specificity of protein-DNA recognition. The quantification of specificity has enabled us to establish the structure-function analysis of transcription factors, such as the effects of binding cooperativity on target recognition. The method is also applied to real genome sequences, predicting potential target sites. We are also using computer simulations of protein-DNA interactions and DNA conformation in order to complement the empirical method. The integration of these approaches together will provide deeper insight into the mechanism of protein-DNA recognition and improve the target prediction of transcription factors.

Binding Sites↗
PubMed1993

Conservation of genetic information: a code for site-specific DNA recognition.

We present findings of genetic information conservation between the glucocorticoid response element (GRE) DNA and the cDNA encoding the glucocorticoid receptor (GR) DNA-binding domain (DBD). The regions of nucleotide sub-sequence similarity to the GRE in the GR DBD occur specifically at nucleotide sequences on the ends of exons 3,4, and 5 at their splice junction sites. These sequences encode the DNA recognition helix on exon 3, a beta-strand on exon 4, and a putative alpha-helix on exon 5, respectively. The nucleotide sequence of exon 5 that encodes the putative alpha-helix located on the carboxyl terminus of the GR DBD shares sequence similarity with the flanking nucleotide regions of the GRE. We generated a computer model of the GR DBD using atomic coordinates derived from nuclear magnetic resonance spectroscopy to which we attached the exon 5-encoded putative alpha-helix. We docked this GR DBD structure at the 39-base-pair nucleotide sequence containing the GRE binding site and flanking nucleotides, which contained conserved genetic information. We observed that amino acids of the DNA recognition helix, the beta-strand, and the putative alpha-helix are spatially aligned with trinucleotides identical to their cognate codons within the GRE and its flanking nucleotides.

Amino Acid Sequence↗
PubMed1998

Phosphorylation of thyroid hormone receptors by protein kinase A regulates DNA recognition by specific inhibition of receptor monomer binding.

Thyroid hormone receptor (T3R) alpha-1 and its oncogenic derivative, the v-ERB A protein, are phosphorylated by cAMP-dependent protein kinase A. Although this phosphorylation appears to be necessary for the oncogenic properties of v-ERB A, the mechanism by which phosphorylation influences the functions of v-ERB A and of the normal T3R has not been established. The protein kinase A phosphorylation site in T3Ralpha-1 is within a domain that is known to contribute to the DNA recognition properties of these receptors. We therefore analyzed the effects of protein kinase A phosphorylation on DNA recognition by the normal T3Ralpha and by the v-ERB A oncoprotein. We report here that phosphorylation of these receptor derivatives does not significantly alter the overall affinity of receptor dimers for DNA. However, phosphorylation does notably alter DNA recognition by preventing, or greatly inhibiting, the ability of these receptors to bind to DNA as protein monomers. These studies suggest that the phosphorylation of T3Ralpha-1 and v-ERB A by protein kinase A may provide a means of altering promoter recognition through a post-translational modification.

Amino Acid Sequence↗
PubMed2000

Structure of HAP1-PC7 bound to DNA: implications for DNA recognition and allosteric effects of DNA-binding on transcriptional activation.

HAP1 is a transcription factor in yeast whose DNA-binding domain has been implicated in directly affecting transcriptional activation. Two separate mutations in the DNA-binding domain, S63G (HAP1-PC7) and S63R (HAP1-18), retain wild-type binding affinity. However, HAP1-PC7 is transcriptionally silent while HAP1-18 shows highly elevated levels of transcription. We have determined the X-ray crystal structure of the DNA-binding domain of HAP1-PC7 bound to its DNA target, UAS(CYC7), and compared it to the previously solved HAP1-wt and HAP1-18 complexes to UAS(CYC7). Additionally, we have quantitatively compared the DNA-binding affinity and specificity of the HAP1-PC7, HAP1-18 and HAP1-wt DNA-binding domains. We show that, although the DNA-binding domains of these three proteins bind UAS(CYC7) with comparable affinity and specificity, the protein-DNA interactions are dramatically different between the three complexes. Conserved protein-DNA interactions are largely restricted to an internal DNA sequence that excludes one of the two conserved DNA half-sites of UAS(CYC7) suggesting a mode of recognition distinct from other HAP1 family members. Alternative protein-DNA interactions result in divergent DNA configurations between the three complexes. These results suggest that the differential transcriptional activities of the HAP1, HAP1-18 and HAP1-PC7 proteins are due, at least in part, to alternative protein-DNA contacts, and implies that HAP1-DNA interactions have direct allosteric effects on transcriptional activation.

Allosteric Regulation↗
PubMed1997

Plasticity of tetramer formation by retinoid X receptors. An alternative paradigm for DNA recognition.

Retinoid X receptors (RXRs) are transcription factors that traditionally have been thought to bind DNA as protein dimers. Recently, however, it has been recognized that RXRs can also bind to DNA as protein tetramers. Receptor tetramers form cooperatively on response elements containing suitably reiterated half-sites, and play an important role in determining the specificity of DNA recognition by different nuclear receptors. We report here that RXR tetramers exhibit significant functional plasticity, and form on response elements possessing diverse half-site orientations and spacings. This ability of RXRs to form tetramers and related oligomers appears to contribute to the synergistic transcriptional activation observed when multiple, spatially separated response elements are introduced into a single promoter. Oligomerization may therefore be a common paradigm for DNA recognition and combinatorial regulation by several different classes of transcription factors.

Animals↗
PubMed2000

DNA recognition by quinoline antibiotics: use of base-modified DNA molecules to investigate determinants of sequence-specific binding of luzopeptin.

The luzopeptin antibiotics contain a cyclic decadepsipeptide to which are attached two quinoline chromophores that bisintercalate into DNA. Although they bind DNA less tightly than the structurally related quinoxaline antibiotics echinomycin and triostin A, the molecular basis of their interaction remains unclear. We have used the PCR in conjunction with novel nucleotides to create specifically modified DNA for footprinting experiments. In order to study the influence that removal, addition or relocation of the guanine 2-amino group, which normally identifies G.C base pairs from the minor groove, has on the interaction of luzopeptin antibiotics with DNA. The presence of a purine 2-amino group is not strictly required for binding of luzopeptin to DNA, but the exact location of this group can alter the position of preferred drug binding sites. It is, however, not the sole determinant of nucleotide sequence recognition in luzopeptin-DNA interaction. Nor can the selectivity of luzopeptin be attributed to the quinoline chromophores, suggesting that an analogue mode of DNA recognition may be operative. This is in contrast to the digital readout that seems to predominate with the quinoxaline antibiotics.

2-Aminopurine↗
PubMed1996

The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation.

The DNA-binding domain of Myb consists of three imperfect repeats, R1, R2 and R3, each containing a helix-turn-helix motif variation. Among these repeats, R2 has distinct characteristics with high thermal instability. The NMR structure analysis found a cavity inside the hydrophobic core of R2 but not in R1 or R3. Here, we show that R2 has slow conformational fluctuations, and that a cavity-filling mutation which stabilizes the R2 structure significantly reduces specific Myb DNA-binding activity and trans-activation. Structural observations of the free and DNA-complexed stages suggest that the implied inherent conformational flexibility of R2, associated with the presence of the cavity, could be important for DNA recognition by Myb.

Amino Acid Sequence↗
PubMed2002

Specificity of protein-DNA recognition revealed by structure-based potentials: symmetric/asymmetric and cognate/non-cognate binding.

Asymmetric binding of protein homodimers to DNA, which has been observed in a number of protein-DNA complexes, leads to subtle structural differences between the two subunits. Such structural differences are frequently observed when the subunits form cognate and non-cognate protein-DNA complexes, respectively. Analysis of these structural effects on binding specificity should provide insight into the mechanism of protein-DNA recognition. We previously derived empirical potential functions for specific nucleotide base-amino acid interactions from statistical analyses of the structures of many protein-DNA complexes and used a combinatorial threading procedure to evaluate the fitness of the DNA sequences involved. We then introduced Z-scores to measure the specificity with which proteins bind to DNA within complexes, as compared to random DNA sequences. Here, we examined in detail the structural effects of asymmetric and cognate/non-cognate binding on specificity. Marked differences in the specificity of DNA binding were observed for the two subunits of lambda repressor, the glucocorticoid receptor, and for transcription factors containing a Zn(2)Cys(6) binuclear cluster domain, which are known to bind asymmetrically to DNA. Moreover, the differences in the specificity with which BamH1 and EcoRV endonucleases bind to their cognate and non-cognate DNA sequences were clearly detected using this approach; indeed, analysis of EcoRV binding enabled us to show the cooperative effect of sequence and structure on binding specificity. The present results demonstrate the utility of this approach when examining the structure-specificity relationship in protein-DNA recognition, as subtle structural differences in symmetric/asymmetric and cognate/non-cognate binding were clearly shown to cause marked differences in specificity. This method can also be used as a tool for checking new structures of protein-DNA complexes for their specificity.

DNA↗
PubMed1997

The SRY cantilever motif discriminates between sequence- and structure-specific DNA recognition: alanine mutagenesis of an HMG box.

The high-mobility-group (HMG) box defines a DNA-bending motif conserved among architectural transcription factors. A "hydrophobic wedge" at the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain "cantilever." First described in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the crux of the HMG box (residues 12 and 13). Here, the role of these side chains in DNA recognition is investigated by alanine mutagenesis. F12A and I13A substitutions in the SRY HMG box each permit native folding and thermal stability (as monitored by circular dichroism and 1H-NMR) but eliminate sequence-specific DNA-binding activity (as detected by gel-mobility shift). On binding to the sharp angles of a four-way DNA junction (4WJ), however, the substitutions each promote formation of a high-molecular-weight aggregate, presumably by DNA-dependent oligomerization. The substitutions have opposite effects on initial binding to the 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enhanced by I13A. A foreshortened "alanine cantilever", not observed among specific HMG boxes, occurs in a non-specific domain (HMG-1A) and may enhance architecture-selective DNA recognition.

Alanine↗
PubMed2005

Sequence-dependent conformational energy of DNA derived from molecular dynamics simulations: toward understanding the indirect readout mechanism in protein-DNA recognition.

Sequence dependence of DNA conformation plays a crucial role in its recognition by proteins and ligands. To clarify the relationship between sequence and conformation, it is necessary to quantify the conformational energy and specificity of DNA. Here, we make a systematic analysis of dodecamer DNA structures including all the 136 unique tetranucleotide sequences at the center by molecular dynamics simulations. Using a simplified conformational model with six parameters to describe the geometry of adjacent base pairs and harmonic potentials along these coordinates, we estimated the equilibrium conformational parameters and the harmonic potentials of mean force for the central base-pair steps from many trajectories of the simulations. This enabled us to estimate the conformational energy and the specificity for any given DNA sequence and structure. We tested our method by using sequence-structure threading to estimate the conformational energy and the Z-score as a measure of specificity for many B-DNA and A-DNA crystal structures. The average Z-scores were negative for both kinds of structures, indicating that the potential of mean force from the simulation is capable of predicting sequence specificity for the crystal structures and that it may be used to study the sequence specificity of both types of DNA. We also estimated the positional distribution of conformational energy and Z-score within DNA and showed that they are strongly position dependent. This analysis enabled us to identify particular conformations responsible for the specificity. The presented results will provide an insight into the mechanisms of DNA sequence recognition by proteins and ligands.

Base Sequence↗
PubMed2006

Structural basis for DNA recognition and processing by UvrB.

DNA-damage recognition in the nucleotide excision repair (NER) cascade is a complex process, operating on a wide variety of damages. UvrB is the central component in prokaryotic NER, directly involved in DNA-damage recognition and guiding the DNA through repair synthesis. We report the first structure of a UvrB-double-stranded DNA complex, providing insights into the mechanism by which UvrB binds DNA, leading to formation of the preincision complex. One DNA strand, containing a 3' overhang, threads behind a beta-hairpin motif of UvrB, indicating that this motif inserts between the strands of the double helix, thereby locking down either the damaged or undamaged strand. The nucleotide directly behind the beta-hairpin is flipped out and inserted into a small, highly conserved pocket in UvrB.

Bacillus↗
PubMed1994

DNA sequence specificity of the v-erb A oncoprotein/thyroid hormone receptor: role of the P-box and its interaction with more N-terminal determinants of DNA recognition.

The viral erb A oncogene is a mutated allele of a normal cell gene for a thyroid hormone receptor. The DNA recognition properties of the v-erb A protein are altered from those of the thyroid hormone receptor, due in part to a point mutation in the P-box of the zinc-finger domain of the viral allele. We report here the effects of systematically varying this P-box codon; our results suggest that this P-box amino acid contributes to DNA specificity not by promoting recognition of the appropriate response elements, but rather by excluding binding of the erb A protein to inappropriate half-sites. In this manner, DNA recognition by the v-erb A protein appears to differ from that by the glucocorticoid receptor. A variety of P-box amino acids were compatible with recognition of the prototypic AGGTCA half-site; intriguingly, several of these mutant erb A proteins could also recognize a variety of alternative half-site sequences. Recognition of these alternative half-sites required a compatible amino acid sequence in the N terminus of the erb A protein. Our results begin to define a code by which the identity of the amino acids in the zinc-finger and N-terminal domains is reflected in the DNA recognition properties of the receptor.

Amino Acid Sequence↗
PubMed1999

Specific DNA recognition by F Factor TraY involves beta-sheet residues.

The F Factor TraY protein is a sequence-specific DNA-binding protein required for efficient conjugal transfer. Genetic and biochemical studies indicate that TraY has two functional roles in conjugation. TraY binds to the PY promoter to up-regulate transcription of tra genes. TraY also binds to the plasmid origin of transfer (oriT), serving as an accessory protein in the nicking of F Factor in preparation for transfer. TraY is thought to belong to the ribbon-helix-helix family of transcription factors. These proteins contact DNA using residues of an antiparallel beta-sheet. We engineered and characterized six TraY mutants each having a single potential beta-sheet DNA contact residue replaced with Ala. Most TraY mutants had significantly reduced affinity for the TraY oriT binding site while possessing near wild-type stability and nonspecific DNA recognition. These results indicate that TraY beta-sheet residues participate in DNA recognition, and support inclusion of TraY in the ribbon-helix-helix family.

Amino Acid Sequence↗
PubMed2000

Three-dimensional structural views of damaged-DNA recognition: T4 endonuclease V, E. coli Vsr protein, and human nucleotide excision repair factor XPA.

Genetic information is frequently disturbed by introduction of modified or mismatch bases into duplex DNA, and hence all organisms contain DNA repair systems to restore normal genetic information by removing such damaged bases or nucleotides and replacing them by correct ones. The understanding of this repair mechanism is a central subject in cell biology. This review focuses on the three-dimensional structural views of damaged DNA recognition by three proteins. The first protein is T4 endonuclease V (T4 endo V), which catalyzes the first reaction step of the excision repair pathway to remove pyrimidine-dimers (PD) produced within duplex DNA by UV irradiation. The crystal structure of this enzyme complexed with DNA containing a thymidine-dimer provided the first direct view of DNA lesion recognition by a repair enzyme, indicating that the DNA kink coupled with base flipping-out is important for damaged DNA recognition. The second is very short patch repair (Vsr) endonuclease, which recognizes a TG mismatch within the five base pair consensus sequence. The crystal structure of this enzyme in complex with duplex DNA containing a TG mismatch revealed a novel mismatch base pair recognition scheme, where three aromatic residues intercalate from the major groove into the DNA to strikingly deform the base pair stacking but the base flipping-out does not occur. The third is human nucleotide excision repair (NER) factor XPA, which is a major component of a large protein complex. This protein has been shown to bind preferentially to UV- or chemical carcinogen-damaged DNA. The solution structure of the XPA central domain, essential for the interaction of damaged DNA, was determined by NMR. This domain was found to be divided mainly into a (Cys)4-type zinc-finger motif subdomain for replication protein A (RPA) recognition and the carboxyl terminal subdomain responsible for DNA binding.

Amino Acid Motifs↗
PubMed1994

DNA recognition by splicing variants of the Wilms' tumor suppressor, WT1.

The Wilms' tumor suppressor, WT1, is a zinc finger transcriptional regulator which exists as multiple forms owing to alternative mRNA splicing. The most abundant splicing variants contain a nine-nucleotide insertion encoding lysine, threonine, and serine (KTS) in the H-C link region between the third and fourth WT1 zinc fingers which disrupts binding to a previously defined WT1-EGR1 binding site. We have identified WT1[+KTS] binding sites in the insulin-like growth factor II gene and show that WT1[+KTS] represses transcription from the insulin-like growth factor II P3 promoter. The highest affinity WT1[+KTS] DNA binding sites included nucleotide contacts involving all four WT1 zinc fingers. We also found that different subsets of three WT1 zinc fingers could bind to distinct DNA recognition elements. A tumor-associated, WT1 finger 3 deletion mutant was shown to bind to juxtaposed nucleotide triplets for the remaining zinc fingers 1, 2, and 4. The characterization of novel WT1 DNA recognition elements adds a new level of complexity to the potential gene regulatory activity of WT1. The results also present the possibility that altered DNA recognition by the dominant WT1 zinc finger 3 deletion mutant may contribute to tumorigenesis.

Alternative Splicing↗