Mechanism Of DNA Recognition Research Articles

Crystal Structures of Multiple GATA Zinc Fingers Bound to DNA Reveal New Insights into DNA Recognition and Self-Association by GATA

The GATA family of transcription factors (GATA1–6) binds selected GATA sites in vertebrate genomes to regulate specific gene expression. Although vertebrate GATA factors have two highly conserved zinc finger motifs, how the two fingers act together to recognize functional DNA elements is not well understood. Here we determined the crystal structures of the C-terminal zinc finger of mouse GATA3 bound to DNA containing two variously arranged GATA binding sites. Our structures and accompanying biochemical analyses reveal two distinct modes of DNA binding by GATA to closely arranged sites. One mode involves cooperative binding by two GATA factors that interact with each other through protein–protein interactions. The other involves simultaneous binding of the N-terminal zinc finger (N-finger) and the C-terminal zinc finger of the same GATA factor. Our studies represent the first crystallographic analysis of GATA zinc fingers bound to DNA and provide new insights into the DNA recognition mechanism by the GATA zinc finger. Our crystal structure also reveals a dimerization interface in GATA that has previously been shown to be important for GATA self-association. These findings significantly advance our understanding of the structure and function of GATA and provide an important framework for further investigating the in vivo mechanisms of GATA-dependent gene regulation.

Structure/function relationships underlying regulation of FOXO transcription factors

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein-protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein-protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.

TINY, a Dehydration-responsive Element (DRE)-binding Protein-like Transcription Factor Connecting the DRE- and Ethylene-responsive Element-mediated Signaling Pathways in Arabidopsis

Dehydration-responsive element-binding proteins (DREBs) and ethylene-responsive element (ERE) binding factors are two major subfamilies of the AP2/ethylene-responsive element-binding protein family and play crucial roles in the regulation of abiotic- and biotic-stress responses, respectively. In the present work, we have reported a previously identified DREB-like factor, TINY, that was involved in both abiotic- and biotic-stress signaling pathways. TINY was capable of binding to both DRE and ERE with similar affinity and could activate the expression of reporter genes driven by either of these two elements in tobacco cells. The 15th amino acid in the APETALA2 (AP2)/ethylene-responsive element-binding factor domain was demonstrated to be essential for its specific binding to ERE, whereas the 14th and 19th amino acids were responsible for the binding to DRE. The expression of TINY was greatly activated by drought, cold, ethylene, and slightly by methyl jasmonate. Additionally, overexpression of TINY in Arabidopsis resulted in elevated expressions of both the DRE- and the ERE-containing genes. Moreover, the expression of DRE-regulated genes, such as COR6.6 and ERD10, was up-regulated upon ethylene treatment, and the expression of ERE-regulated genes, such as HLS1, was also increased by cold stress, when the expression of TINY was being induced. These results strongly suggested that TINY might play a role in the cross-talk between abiotic- and biotic-stress-responsive gene expressions by connecting the DRE- and ERE-mediated signaling pathways. The results herein might promote the understanding of the mechanisms of specific DNA recognition and gene expression regulation by DREBs.

Structural studies of RecF

RecF, together with RecO and RecR, belong to a ubiquitous group of recombination mediators (RMs) that include eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA‐like recombinases and displacing single‐stranded DNA binding proteins. Here we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled‐coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so‐called “Lobe II” subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double‐stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP‐dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

Neutrophil recognition of bacterial DNA and Toll-like receptor 9-dependent and -independent regulation of neutrophil function

Neutrophils are essential for host defense and detect the presence of invading microorganisms through recognition of pathogen-associated molecular patterns. Among these receptors are Toll-like receptors (TLRs). Neutrophils express all known TLRs except for TLR3. TLR9, localized intracellularly, is to date the best characterized sensor for bacterial DNA, containing short sequences of unmethylated CpG motifs, though TLR9-independent intracellular DNA recognition mechanism(s) may also exist. Bacterial DNA has profound impact on neutrophil functions; it promotes neutrophil trafficking in vivo, induces chemokine expression, regulates expression of adhesion molecules, enhances phagocyte activity, and rescues neutrophils from constitutive apoptosis. TLR9 stimulation results in alterations in cellular redox balance, peroxynitrite formation, activation of the mitogen-activated protein kinase, PI3-kinase, and Jun N-terminal kinase pathways and/or nuclear factor kappaB and AP-1. These features identify an important role for bacterial DNA and TLR9 signaling in the regulation of neutrophil functions that are critical for optimal expression as well as for resolution of the inflammatory response.

The papillomavirus E2 DNA binding domain

The DNA binding domain of the E2 master regulator from papillomaviruses is the primary effector for most the essential activities controlled by this protein. In this review we focus on the properties of the DNA binding domain of human papillomavirus strain 16 in solution, integrating structure, dynamics, folding, stability, conformational equilibria, and DNA binding mechanism. We discuss the relevance of these processes for the different biological activities, broadening the horizon for antiviral development. In addition, the particular fold of the DNA binding domain only shared with the Epstein-Barr nuclear antigen EBNA1, suggests a link between this unique architecture and the function of viral origin binding proteins of this kind. Finally, the E2 DNA binding domain proved to be an excellent model for addressing fundamental problems of DNA recognition mechanisms and folding of intertwined dimers.

DNA sensors in innate immune system

Microbial sensing mediated by pattern recognition receptors (PRRs) is the first key step to trigger innate immune responses, represented by the induction of type I interferons (IFNs), proinflammatory cytokines and chemokines. This innate signaling elicits an efficient activation of more specific responses in adaptive immunity. Such coordinated responses in the two systems are essential for the optimal elimination of invading microbes. Despite a major advance in our understanding of RNA sensors, TLR9 remained the only known sensor of DNA. On the other hand, there has been accumulating evidence supporting the existence of TLR9-independent DNA recognition mechanism. In this regard, DAI (also termed as DLM-1/ZBP1), the first sensor of cytosolic DNA, has recently been identified with its activation of IFN-regulatory factors(IRFs) and NF-kappaB transcriptional factors. Several recent papers suggest the involvement of an additional cytosolic DNA sensor(s). There is also a recent report that cytosolic microbial and host DNA can trigger pro-inflammatory responses via the TLR- and IRF-indepnedent pathway mediated by the inflammasome, which is consisted of NLR family members together with the adaptor protein ASC and caspase-1. In addition, evidence has been provided that host- and virus-derived proteins, which contain DNA-binding motifs (Zalpha and/or Zbeta) similar to those of DAI(DLM-1/ZBP1), negatively regulates the immune response that is activated by cytosolic DNA. Thus, these recent findings reveal the complex DNA-sensing mechanism for triggering the activation of innate immunity, and the breakdown of this sensing mechanism may lead to autoimmune abnormalities.

Functional Characterization of the Escherichia coli Fis–DNA Binding Sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis–DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis–DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis–DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C 5 methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis–DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level.

The M.EcoRV DNA-(Adenine N6)-methyltransferase Uses DNA Bending for Recognition of an Expanded EcoDam Recognition Site

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys11 of M.EcoRV is involved in recognition of Gua1 and Arg128 contacts the Gua in base pair 6. These residues correspond to Lys9 and Arg124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59+/-4 degrees) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua6-Arg128 contact forms early followed by DNA bending and, finally, the formation of the Lys11-Gua1 contact.

The prokaryotic Cys 2 His 2 zinc-finger adopts a novel fold as revealed by the NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain

The first putative prokaryotic Cys(2)His(2) zinc-finger domain has been identified in the transcriptional regulator Ros from Agrobacterium tumefaciens, indicating that the Cys(2)His(2) zinc-finger domain, originally thought to be confined to the eukaryotic kingdom, could be widespread throughout the living kingdom from eukaryotic, both animal and plant, to prokaryotic. In this article we report the NMR solution structure of Ros DNA-binding domain (Ros87), providing 79 structural characterization of a prokaryotic Cys(2)His(2) zinc-finger domain. The NMR structure of Ros87 shows that the putative prokaryotic Cys(2)His(2) zinc-finger sequence is indeed part of a significantly larger zinc-binding globular domain that possesses a novel protein fold very different from the classical fold reported for the eukaryotic classical zinc-finger. The Ros87 globular domain consists of 58 aa (residues 9-66), is arranged in a betabetabetaalphaalpha topology, and is stabilized by an extensive 15-residue hydrophobic core. A backbone dynamics study of Ros87, based on (15)N R(1), (15)N R(2), and heteronuclear (15)N-{(1)H}-NOE measurements, has further confirmed that the globular domain is uniformly rigid and flanked by two flexible tails. Mapping of the amino acids necessary for the DNA binding onto Ros87 structure reveals the protein surface involved in the DNA recognition mechanism of this new zinc-binding protein domain.

Probing of contacts between EcoRII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.

BAFfled by Poxviruses?

Successful viruses engage in a dynamic interplay with their hosts, where both utilize diverse strategies to impose their supremacy. In this issue of Cell Host & Microbe, Wiebe and Traktman describe a novel interaction between vaccinia virus and mammalian cells. A host protein called BAF can bind ectopic cytoplasmic DNA and block viral DNA replication, whereas vaccinia in turn counteracts this inhibition with a virus-encoded serine threonine kinase that inactivates BAF.

Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function

RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called 'Lobe II' subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites.

Studies of DNA recognition mechanism of transcription factor IRF-4

Transcription factor IFN regulatory factor-4 (IRF-4) prefers a DNA sequence including CCGAAA, though the consensus DNA-binding sequence of the IRF family proteins is NNGAAA, and the crystal structure of PU.1/IRF-4/DNA (GTGAAA) ternary complex indicates the NN region of DNA does not interact with IRF-4 directly. This suggests that there is an indirect DNA recognition mechanism in IRF-4. In order to account for the sequence preference of IRF-4, we focused on structural properties of DNA duplexes recognized by IRF-4. Here, we performed solution NMR studies on DNA duplexes containing GGGAAA and CCGAAA sequences, and assigned most of proton resonances of DNA 17 mer with GGGAAA. (1)H-(1)H NOESY spectra indicated B-form like structure for GGGAAA. We also assigned imino proton resonances of DNA 17 mer with CCGAAA. For the imino proton region, the (1)H-(1)H NOESY spectra of these two DNA duplexes were similar.

Further Insight into the DNA Recognition Mechanism of Trabectedin from the Differential Affinity of Its Demethylated Analogue Ecteinascidin ET729 for the Triplet DNA Binding Site CGA

Trabectedin and its N12-demethylated analogue ET729 bind covalently to the central guanine of selected DNA triplets. Although both drugs equally target several sites, including AGA, we show that covalent modification of CGA is only achieved by ET729. By means of molecular dynamics simulations of the precovalent complexes, we explain in atomic detail how such a simple structural modification brings about this notable change in the DNA-binding selectivity profiles of these two drugs.

Mechanism of DNA Recognition at a Viral Replication Origin

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.

Structure of the Forkhead Domain of FOXP2 Bound to DNA

FOXP (FOXP1-4) is a newly defined subfamily of the forkhead box (FOX) transcription factors. A mutation in the FOXP2 forkhead domain cosegregates with a severe speech disorder, whereas several mutations in the FOXP3 forkhead domain are linked to the IPEX syndrome in human and a similar autoimmune phenotype in mice. Here we report a 1.9 A crystal structure of the forkhead domain of human FOXP2 bound to DNA. This structure allows us to revise the previously proposed DNA recognition mechanism and provide a unifying model of DNA binding for the FOX family of proteins. Our studies also reveal that the FOXP2 forkhead domain can form a domain-swapped dimer, made possible by a strategic substitution of a highly conserved proline in conventional FOX proteins with alanine in the P subfamily. Disease-causing mutations in FOXP2 and FOXP3 map either to the DNA binding surface or the domain-swapping dimer interface, functionally corroborating the crystal structure.

Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains

Type II restriction endonuclease Mva1269I recognizes an asymmetric DNA sequence 5'-GAATGCN / -3'/5'-NG / CATTC-3' and cuts top and bottom DNA strands at positions, indicated by the "/" symbol. Most restriction endonucleases require dimerization to cleave both strands of DNA. We found that Mva1269I is a monomer both in solution and upon binding of cognate DNA. Protein fold-recognition analysis revealed that Mva1269I comprises two "PD-(D/E)XK" domains. The N-terminal domain is related to the 5'-GAATTC-3'-specific restriction endonuclease EcoRI, whereas the C-terminal one resembles the nonspecific nuclease domain of restriction endonuclease FokI. Inactivation of the C-terminal catalytic site transformed Mva1269I into a very active bottom strand-nicking enzyme, whereas mutants in the N-terminal domain nicked the top strand, but only at elevated enzyme concentrations. We found that the cleavage of the bottom strand is a prerequisite for the cleavage of the top strand. We suggest that Mva1269I evolved the ability to recognize and to cleave its asymmetrical target by a fusion of an EcoRI-like domain, which incises the bottom strand within the target, and a FokI-like domain that completes the cleavage within the nonspecific region outside the target sequence. Our results have implications for the molecular evolution of restriction endonucleases, as well as for perspectives of engineering new restriction and nicking enzymes with asymmetric target sites.

Functional Identification of an 8-Oxoguanine Specific Endonuclease from Thermotoga maritima

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of NaBH4, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.