Sequence-specific DNA Recognition Research Articles

1H, 13C and 15N chemical shift assignments for the human Pitx2 homeodomain and a R24H homeodomain mutant

The homeodomain is one of the most important eukaryotic DNA-binding motifs and has been identified in over one thousand proteins. Homeodomain proteins play critical roles in diverse biological processes, including cell differentiation and cell pattern formation. The human Pitx2 homeodomain binds several different DNA sequences and is a pivotal component of both the TGF-β and Wnt/β-catenin signaling pathways. As the recognition of specific DNA sequences represents an essential biochemical function of all DNA-binding proteins, we have chosen the Pitx2 homeodomain model to investigate the mechanisms that convey biological specificity in these protein-DNA interactions. Here, we report complete chemical shift assignments of the human Pitx2 homeodomain and the R24H mutation that induces ring dermoid of the cornea syndrome.

Sequence-specific recognition of methylated DNA by human zinc-finger proteins

DNA methylation is an essential epigenetic mark. Three classes of mammalian proteins recognize methylated DNA: MBD proteins, SRA proteins and the zinc-finger proteins Kaiso, ZBTB4 and ZBTB38. The last three proteins can bind either methylated DNA or unmethylated consensus sequences; how this is achieved is largely unclear. Here, we report that the human zinc-finger proteins Kaiso, ZBTB4 and ZBTB38 can bind methylated DNA in a sequence-specific manner, and that they may use a mode of binding common to other zinc-finger proteins. This suggests that many other sequence-specific methyl binding proteins may exist.

新規遺伝子発現制御化合物としてのピロールポリアミド-ヌクレオシドハイブリッド及びそのハイブリッドを組込んだオリゴマーの設計，合成及び評価

On the basis of reports that a minor groove binder pyrrolepolyamide can interfere with gene expression by the sequence-specific recognition of DNA, we expected that nucleoside bearing a pyrrolepolyamide would be able to regulate gene expression. Therefore, we designed and synthesized the pyrrolepolyamide-adenosine (Hybrid 1) and -2'-deoxyguanosine hybrids (Hybrid 2 and Hybrid 3) as lead compounds for gene expression control compounds. The pyrrolepolyamide frame of Hybrid 2 and Hybrid 3 combines at the 2-exocyclic amino group of the 2'-deoxyguanosine by a linker and the 2-exocyclic amino group of guanine exists in the minor groove side of the duplex. Hybrid 2 is the 2'-deoxyguanosine-pyrrolepolyamide hybrid using the 3-aminopropionyl linker, while Hybrid 3 uses the 3-aminopropyl linker. An evaluation of the DNA binding sequence selectivity was performed by analysis of T(m) values and CD spectra, using distamycin A as a contrast. Hybrid 3 has provided more excellent sequence-distinguishable ability than other hybrids and Distamycin A. Moreover, on the basis of these results, we synthesized oligonucleotides conjugated to Hybrid 4, which is stable under conditions of DNA oligonucleotide solid phase synthesis, arranged from Hybrid 3. From T(m) values and CD spectral analysis, it was found that oligonucleotides conjugating Hybrid 4 possess high recognition ability and very high binding ability for the DNA that includes the pyrrolepolyamide binding sequence.

THAP proteins target specific DNA sites through bipartite recognition of adjacent major and minor grooves.

THAP-family C2CH zinc-coordinating DNA-binding proteins function in diverse eukaryotic cellular processes, such as transposition, transcriptional repression, stem-cell pluripotency, angiogenesis and neurological function. To determine the molecular basis for sequence-specific DNA recognition by THAP proteins, we solved the crystal structure of the Drosophila melanogaster P element transposase THAP domain (DmTHAP) complexed with a natural 10-base pair site. In contrast to C2H2 zinc fingers, DmTHAP docks a conserved β-sheet into the major groove and a basic C-terminal loop into the adjacent minor groove. We confirmed specific protein-DNA interactions by mutagenesis and DNA binding assays. Sequence analysis of natural and in-vitro-selected binding sites suggests several THAPs (DmTHAP, human THAP1 and THAP9) recognize a bipartite TxxGGGx(A/T) consensus motif; homology suggests THAP proteins bind DNA through a bipartite interaction. These findings reveal the conserved mechanisms by which THAP-family proteins engage specific chromosomal target elements.

The role of DNA shape in protein–DNA recognition

The recognition of specific DNA sequences by proteins is thought to depend on two types of mechanisms: one that involves the formation of hydrogen bonds with specific bases, primarily in the major groove, and one involving sequence-dependent deformations of the DNA helix. By comprehensively analyzing the three dimensional structures of protein-DNA complexes, we show that the binding of arginines to narrow minor grooves is a widely used mode for protein-DNA recognition. This readout mechanism exploits the phenomenon that narrow minor grooves strongly enhance the negative electrostatic potential of the DNA. The nucleosome core particle offers a striking example of this effect. Minor groove narrowing is often associated with the presence of A-tracts, AT-rich sequences that exclude the flexible TpA step. These findings suggest that the ability to detect local variations in DNA shape and electrostatic potential is a general mechanism that enables proteins to use information in the minor groove, which otherwise offers few opportunities for the formation of base-specific hydrogen bonds, to achieve DNA binding specificity.

Sequence-specific Recognition of DNA by the C-terminal Domain of Nucleoid-associated Protein H-NS

The molecular determinants necessary and sufficient for recognition of its specific DNA target are contained in the C-terminal domain (H-NSctd) of nucleoid-associated protein H-NS. H-NSctd protects from DNaseI cleavage a few short DNA segments of the H-NS-sensitive hns promoter whose sequences closely match the recently identified H-NS consensus motif (tCG(t/a)T(a/t)AATT) and, alone or fused to the protein oligomerization domain of phage lambda CI repressor, inhibits transcription from the hns promoter in vitro and in vivo. The importance of H-NS oligomerization is indicated by the fact that with an extended hns promoter construct (400 bp), which allows protein oligomerization, DNA binding and transcriptional repression are highly and almost equally efficient with native H-NS and H-NSctd::lambdaCI and much less effective with the monomeric H-NSctd. With a shorter (110 bp) construct, which does not sustain extensive protein oligomerization, transcriptional repression is less effective, but native H-NS, H-NSctd::lambdaCI, and monomeric H-NSctd have comparable activity on this construct. The specific H-NS-DNA interaction was investigated by NMR spectroscopy using monomeric H-NSctd and short DNA duplexes encompassing the H-NS target sequence of hns (TCCTTACATT) with the best fit (8 of 10 residues) to the H-NS-binding motif. H-NSctd binds specifically and with high affinity to the chosen duplexes via an overall electropositive surface involving four residues (Thr(109), Arg(113), Thr(114), and Ala(116)) belonging to the same protein loop and Glu(101). The DNA target is recognized by virtue of its sequence and of a TpA step that confers a structural irregularity to the B-DNA duplex.

Sequence Dependencies of DNA Deformability and Hydration in the Minor Groove

DNA deformability and hydration are both sequence-dependent and are essential in specific DNA sequence recognition by proteins. However, the relationship between the two is not well understood. Here, systematic molecular dynamics simulations of 136 DNA sequences that differ from each other in their central tetramer revealed that sequence dependence of hydration is clearly correlated with that of deformability. We show that this correlation can be illustrated by four typical cases. Most rigid basepair steps are highly likely to form an ordered hydration pattern composed of one water molecule forming a bridge between the bases of distinct strands, but a few exceptions favor another ordered hydration composed of two water molecules forming such a bridge. Steps with medium deformability can display both of these hydration patterns with frequent transition. Highly flexible steps do not have any stable hydration pattern. A detailed picture of this correlation demonstrates that motions of hydration water molecules and DNA bases are tightly coupled with each other at the atomic level. These results contribute to our understanding of the entropic contribution from water molecules in protein or drug binding and could be applied for the purpose of predicting binding sites.

Sequence-specific recognition of DNA by lac-repressor headpiece

Interaction of amino acid sequences Ala—Gln—Gln—Leu—Ala (Sequence 53–57) and Tyr—Gln—Thr—Val—Ser—Arg—Val—Val—Asn (Sequence 17–25) from the lac-repressor headpiece with pentanucleotide duplexes in B-form is studied in the minor and major groove, respectively, by computer-based model building. These peptides were taken in β-antiparallel and α-helical conformations. Our results show that backbone flexibility is essential for the recognition of the peptide in β conformation in the minor groove, whereas for α helix, side-chain flexibility is sufficient for making sequence-specific contacts in the major groove. Occurrence of these two structures provides a suitable recognition code for the repressor headpiece.

Intrinsic Dynamics of Restriction Endonuclease EcoO109I Studied by Molecular Dynamics Simulations and X-Ray Scattering Data Analysis

EcoO109I is a type II restriction endonuclease that functions as a dimer in solution. Upon DNA binding to the enzyme, the two subunits rotate counterclockwise relative to each other, as the two catalytic domains undergo structural changes to capture the cognate DNA. Using a 150-ns molecular dynamics simulation, we investigated the intrinsic dynamics of the DNA-free enzyme in solution to elucidate the relationship between enzyme dynamics and structural changes. The simulation revealed that the enzyme is considerably flexible, and thus exhibits large fluctuations in the radius of gyration. The small-angle x-ray scattering profile calculated from the simulation, including scattering from explicit hydration water, was in agreement with the experimentally observed profile. Principal component analysis revealed that the major dynamics were represented by the open-close and counterclockwise motions: the former is required for the enzyme to access DNA, whereas the latter corresponds to structural changes upon DNA binding. Furthermore, the intrinsic dynamics in the catalytic domains were consistent with motions capturing the cognate DNA. These results indicate that the structure of EcoO109I is intrinsically flexible in the direction of its functional movement, to facilitate effective structural changes for sequence-specific DNA recognition and processing.

DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure−Function Study of the Zinc-Binding Domain

Synthesis of oligoribonucleotide primers for lagging-strand DNA synthesis in the DNA replication system of bacteriophage T7 is catalyzed by the primase domain of the gene 4 helicase-primase. The primase consists of a zinc-binding domain (ZBD) and an RNA polymerase (RPD) domain. The ZBD is responsible for recognition of a specific sequence in the ssDNA template whereas catalytic activity resides in the RPD. The ZBD contains a zinc ion coordinated with four cysteine residues. We have examined the ligation state of the zinc ion by X-ray absorption spectroscopy and biochemical analysis of genetically altered primases. The ZBD of primase engaged in catalysis exhibits considerable asymmetry in coordination to zinc, as evidenced by a gradual increase in electron density of the zinc together with elongation of the zinc-sulfur bonds. Both wild-type primase and primase reconstituted from purified ZBD and RPD have a similar electronic change in the level of the zinc ion as well as the configuration of the ZBD. Single amino acid replacements in the ZBD (H33A and C36S) result in the loss of both zinc binding and its structural integrity. Thus the zinc in the ZBD may act as a charge modulation indicator for the surrounding sulfur atoms necessary for recognition of specific DNA sequences.

Modulation of pKa of Brooker’s Merocyanine by DNA Hybridization

Brooker's merocyanine (BM), which changes its emission and absorption maxima upon protonation, was introduced into oligodeoxyribonucleotide (ODN) via d-threoninol by postsynthetic modification on a CPG (controlled-pore glass) support. The pK(a) of BM in the modified ODN increased from 9.5 to 10.1 upon hybridization. As a result, absorption maxima shifted from 492 to 432 nm at pH 10.0 by the presence of its complementary strand. This spectral shift was sufficiently large so that DNA hybridization could easily be discriminated even by the naked eye; the color of the solution changed from orange to yellow upon hybridization. In addition, the fluorescence emission was strongly quenched upon hybridization, demonstrating that this probe can also detect the target DNA by the fluorescence change. Ratiometric detection of hybridization was also possible by simultaneous excitation of both protonated and deprotonated BMs. Furthermore, we could also modulate its pK(a) by the antiparallel stacking of two BM molecules in the duplex; the pK(a) of BM decreased from 10.1 to 9.7 by the stacking of two BMs in an antiparallel manner. Thus, control of the microenvironment around the BM molecule allowed modulation of its pK(a), which is applicable to the sequence-specific recognition of target DNA.

Predicting the binding preference of transcription factors to individual DNA k-mers

Motivation: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA–protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members.Results: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF–DNA recognition, and suggest a rational approach for future analyses of TF families.Contact: t.hughes@utorotno.caSupplementary information: Supplementary data are available at Bioinformatics online.

RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPγS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2′-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPγS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction.

Epigenomics: A Roadmap, But to Where?

Recently, the Director of the National Institutes of Health (NIH) allocated $190 million for an “Epigenomics” Roadmap initiative ([1][1]). As investigators in this area, we endorse the idea that chromatin biology is an appropriate, if not essential, area for the NIH to support, not only for its fundamental biological significance but also its relevance to human disease. Nonetheless, we believe that this initiative, at least in its current form, will not yield significant benefits. If the use of the term “epigenome” is intended to equate the value of this Roadmap initiative with the Human Genome Project, it fails on several grounds. First, it does not consider our current understanding of the roles of sequence-specific DNA recognition events and transcriptional networks in controlling epigenetic changes. A multifaceted effort that elucidates transcriptional circuits that tell us where and when signal-responsive, sequence-specific regulators function would be more useful for understanding cell type programming. Second, merely cataloging modification patterns offers comparatively little new or useful information. We already know that most genes are associated with one of a few patterns of chromatin modifications and that the patterns themselves do not tell us how that gene is regulated or how its expression state is inherited. Most histone modifications are highly dynamic and change rapidly in response to changes in signals that turn genes on or off. This initiative will divert substantial resources, enough to fund 200 multiyear individual grants. There is a notion favored by some that individual scientists need to be corralled to work together under a more rigid, directed framework to solve important problems. We disagree. Real innovation comes from the bottom up, and good science policy requires promoting the free market of ideas rather than central planning ([2][2]). 1. 1.[↵][3] NIH Roadmap for Medical Research ( ). 2. 2.[↵][4] Links to a full version of this letter and petition for readers to sign can be found at . [1]: #ref-1 [2]: #ref-2 [3]: #xref-ref-1-1 View reference 1. in text [4]: #xref-ref-2-1 View reference 2. in text

Variation in Homeodomain DNA Binding Revealed by High-Resolution Analysis of Sequence Preferences

Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity and showing that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

Identification of a Consensus DNA-Binding Site for the Arabidopsis thaliana SBP Domain Transcription Factor, AtSPL14, and Binding Kinetics by Surface Plasmon Resonance

Proteins with a conserved Cys- and His-rich SQUAMOSA promoter binding protein (SBP) domain are transcription factors restricted to photosynthetic organisms that possess a novel two Zn-finger structure DNA-binding domain. Despite the fact that altered expression of some SBP-encoding genes has profound effects on organism growth and development, little is known about SBP domain protein target genes. Misexpression of the Arabidopsis thaliana AtSPL14 SBP domain gene confers resistance to programmed cell death and modifies plant architecture. A consensus DNA-binding motif for AtSPL14 was identified by systematic evolution of ligands by exponential enrichment (SELEX) or random binding site selection (RBSS). DNA recognized by AtSPL14 contained the core binding motif (GTAC) found for other SBP domain proteins, but mutational analyses indicated that at least one additional flanking nucleotide is necessary for effective AtSPL14-DNA interaction. Comparison of several SBP domain amino acid sequences allows us to hypothesize which specific amino acids might participate in this sequence-specific DNA recognition. Electrophoretic mobility shift assays (EMSA) with mutant AtSPL14 DNA-binding domain proteins indicated that not all of the Zn (2+) ion coordinating ligands in the second Zn structure are strictly required for DNA binding. Surface plasmon resonance (SPR) was used to evaluate AtSPL14 in vitro binding kinetics for comparison of equilibrium binding constants with other SBP domain proteins. These data provide a strong basis for further experiments aimed at defining and distinguishing the sets of genes regulated by the closely related SBP domain family members.

Sequence-Specific Recognition of Double-Stranded DNA by Synthetic Minor Groove Binder Conjugates. toward the Construction of Artificial Site-Specific Deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu 2+ ions was observed.

Performing DNA computation with RecA-mediated triple-stranded DNA structure

A new method of DNA computing using an unusual triple-stranded DNA structure mediated by RecA protein is introduced and a minor 3-variable instance of the satisfiability (SAT) problem is solved. Separations were performed by using biotinylated oligodeoxynucleotide probes coated with RecA protein. During the computation, sequence-specific recognition of double-stranded DNA by homologous oligodeoxynucleotides was fulfilled in the presence of ATPγS. Captured by the magnetic particles coated with streptavidin, the desirable double-stranded DNA strands were retained and extracted. It is suggested that the methods employed here may help decrease the errors in DNA computation and solve some larger size NP- complete problems.

A novel DNA computing model based on RecA-mediated triple-stranded DNA structure

Abstract The field of DNA computing emerged in 1994 after Adleman's paper was published. Henceforth, a few scholars solved some noted NP-complete problems in this way. And all these methods of DNA computing are based on conventional Watson-Click hydrogen bond of doublehelical DNA molecule. In this paper, we show that the triple-stranded DNA structure mediated by RecA protein can be used for solving computational problems. Sequence-specific recognition of double-stranded DNA by oligonucleotide-directed triple helix (triplex) formation is used to carry out the algorithm. We present procedure for the 3-vertex-colorability problems. In our proposed procedure, it is suggested that it is possible to solve more complicated problems with more variables by this model. *Supported by National Natural Science Foundation of China (Grant Nos. 60533010, 60373089, 60674106), Science Foundation of Shaanxi Bureau of Education (Grant No. 06JK202), Program for New Century Excellent Talents in University (No. NCET-05-0612) an...

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.