DNA Contacts Research Articles

Miniature homeodomains: high specificity without an N-terminal arm.

Recently, we described a strategy for the design of miniature proteins that bind DNA and protein surfaces with high affinity and selectivity. This strategy involves identifying the functional epitope required for macromolecular recognition by a natural protein and presenting it on a small, stable protein scaffold. In previous work, high-affinity DNA recognition was achieved only when the miniature protein contained the complete functional epitope. Here we report a miniature homeodomain that recognizes its 6-bp target site in the nanomolar concentration range at 25 degrees C, despite the absence of DNA contact residues located along the homeodomain N-terminal arm. We conclude that miniature proteins can achieve high affinity and selectivity for DNA by design even when the functional epitope is incomplete by using pre-organization to effectively compensate for lost protein-DNA contacts. In this case it has been possible to miniaturize both the recognition surface and the structural framework of a globular protein fold.

Two of the five zinc fingers in the Zap1 transcription factor DNA binding domain dominate site-specific DNA binding.

The Zap1 transcriptional activator from Saccharomyces cerevisiae induces expression of a series of genes containing an 11 base pair conserved promoter element (ZRE) under conditions of zinc deficiency. This work shows that Zap1 uses four of its seven zinc finger domains to contact the ZRE and that two of these dominate the interaction by contacting the essential ACC-GGT ends. Two Zn finger domains (ZF1 and ZF2) do not contact DNA, and a third ZF3 may be more important for interfinger protein-protein interactions. Zn finger domains important for ZRE contact were identified from triple mutations in Zap1, changing three residues in the alpha helix in each finger known to be important for DNA contacts in Zn finger proteins. Replacement of -1, 3, and 6 helix residues in ZF4 and ZF7 reduced the affinity of Zap1 for the wild-type ZRE. In contrast, triple mutations within the intervening ZF5 and ZF6 domains had minimal effect. The data argue that fingers 4 and 7 contact the ACC-GGT ends while fingers 5 and 6 contact the 5 bp central ZRE sequence. This conclusion is corroborated by decreased Zap1 affinity for a ZRE DNA duplex containing mutations of the AC-GT ends of the ZRE, whereas transversion mutations within the central 5 bp of the ZRE had minimal effect on Zap1 binding affinity.

Site-Directed Histone–DNA Contact Mapping for Analysis of Nucleosome Dynamics

This chapter presents methods for site-directed mapping of histone–DNA interactions with single base pair resolution. An experimental approach is devised that allows simultaneous determination of the precise translational position(s) as well as direct structural assessment of the remodeled nucleosome at the level of specific histone–DNA contacts in the regions of the highly conserved histone fold domains. This approach involves selective attachment of photoreactive aryl azide groups to unique cysteines engineered at sites near main interaction points of the nucleosomal DNA with the globular domains of histones H2B, H2A, and H4, respectively, 54, 39, and 2 bp away from the dyad axis of the nucleosome. Determination of the precise points of interaction of these residues with DNA before and after remodeling following UV-induced covalent cross-linking provides an accurate assessment of the distance/path of DNA on the histone octamer surface between six structural reference points (two copies of each of the three residues). Examples of its application are also presented for analysis of the altered translational positioning and structural conformation of nucleosomes remodeled by the SWI/SNF complex.

R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site.

R2 retrotransposons insert into the 28S rRNA genes of insects. Integration occurs by specific cleavage of the target site and utilization of the released DNA end to prime reverse transcription of the RNA transcript. Specificity of the protein to the target site is dependent upon nucleotide sequence recognition extending from 35 bp upstream to 15 bp downstream of the cleavage site. In this report, we show that sequence recognition and cleavage by the R2 protein can occur while the target site is assembled into nucleosomes. Reconstitution of DNA fragments containing the 28S gene sequence into a set of nucleosomes with different translational frames revealed that the R2 site adopted the same rotational orientation with respect to the histone octamer. Binding and cleavage by the R2 protein were most efficient when the upstream binding site for the R2 protein was near a nucleosome end. Interaction of the R2 protein with the nucleosome disrupted the histone:DNA contacts in the 50 bp region directly bound by R2, but did not modify the remainder of the nucleosome structure.

Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes.

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.

High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2.

The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA. The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach. The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed. Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template. In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting. ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding.

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.

The Downstream DNA Jaw of Bacterial RNA Polymerase Facilitates Both Transcriptional Initiation and Pausing

Regulation of RNA polymerase during initiation, elongation, and termination of transcription is mediated in part by interactions with intrinsic regulatory signals encoded in the RNA and DNA that contact the enzyme. These interactions include contacts to an 8-9-bp RNA:DNA hybrid within the active-site cleft of the enzyme, contacts to the melted nontemplate DNA strand in the vicinity of the hybrid, contacts to exiting RNA upstream of the hybrid, and contacts to approximately 20 bp of duplex DNA downstream of the active site. Based on characterization of an amino acid substitution (G1161R) and a deletion (Delta1149-1190) in the jaw domain of the bacterial RNA polymerase largest subunit (beta'), we report here that contacts of the jaw domain to downstream DNA at the leading edge of the transcription complex contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw domain-downstream DNA contact in transcriptional initiation and pausing and suggest possible explanations for the previously reported isolation of the jaw mutants based on reduced ColEI plasmid replication.

Evolution of signalling in the sporulation phosphorelay.

Two-component and phosphorelay signal transduction systems are believed to function as environ-mental sensors that programme gene expression to the composition of the ecological niche in which a microbe normally resides. The question of how evolutionarily related bacteria that occupy different environments change their signal transduction pathways to adapt to such environments was asked of the sporulation phosphorelay of Bacillus subtilis, Bacillus halodurans, Bacillus anthracis and Bacillus stearothermophilus. Comparison of the primary amino acid sequence of phosphorelay proteins with the known structural and interactive properties of the B. subtilis proteins revealed that the amino acid residues of interaction surfaces between phosphorelay proteins and between a phosphorelay protein and DNA resist evolutionary change. The absolute conservation of interaction surfaces allowed the identification of sporulation sensor kinases in B. halodurans, B. anthracis and B. stearothermophilus. In these sensor kinases, the signal-sensing domains are vastly different in size and subdomain composition, with little apparent conservation between species, whereas the catalytic domains of these sensor kinases retain the high level of homology observed for the other phosphorelay proteins. Adaptation to new environments appears to result in rapid evolution of signalling domains to maximize environmental impact while maintaining identical protein-protein and protein-DNA contacts in the entire phosphorelay. In Clostridial genomes, only the Spo0A protein was found, suggesting that the anaerobic relatives of the Bacilli do not use a phosphorelay and phosphorylate Spo0A directly with sensor kinases.

Evidence for "unseen" transposase--DNA contacts.

In this study, evidence of novel, important interactions between a hyperactive Tn5 transposon recognition end sequence and hyperactive Tn5 transposase (Tnp) are presented. A hyperactive Tn5 end sequence, the mosaic end (ME), was isolated previously. The ME and a wild-type end sequence, the outside end (OE), differ at only three positions, yet transposition on the ME is tenfold higher than on the OE in vivo. Also, transposition on the ME is much more efficient than transposition on the OE in vitro. Here, we show that the decreased activity observed for the OE is caused by a defect in paired ends complex (PEC) formation resulting from the orientation of the A-T base-pair at position 4 of this end. Efficient PEC formation requires an interaction between the C5-methyl group (C5-Me) on the non-transferred strand thymine base at position 4 (T4) and Tnp. PEC formation on nicked substrates is much less affected by the orientation of the A-T base-pair at position 4, indicating that the C5-Me group is important only for steps preceding nicking. A recently determined co-crystal structure of Tn5 Tnp with the ME is discussed and a model explaining possible roles for the base-pair at position 4 is explored.

Nucleosome sliding: facts and fiction.

Nucleosome sliding is a frequent result of energy-dependent nucleosome remodelling in vitro. This review discusses the possible roles for nucleosome sliding in the assembly and maintenance of dynamic chromatin and for the regulation of diverse functions in eukaryotic nuclei.

The RUNX1 Runt Domain at 1.25 Å Resolution: A Structural Switch and Specifically Bound Chloride Ions Modulate DNA Binding

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor β, CBFβ, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25 Å resolution and compare its conformation to previously published structures in complex with DNA, CBFβ or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFβ-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFβ alone; suggesting that one role of CBFβ is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding. A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.

Chromatin remodeling by RSC involves ATP-dependent DNA translocation.

Chromatin-remodeling complexes couple ATP hydrolysis to alterations in histone-DNA interactions and nucleosome mobility, allowing transcription factors access to chromatin. Here, we use triple-helix strand-displacement assays, DNA length-dependent ATPase assays, and DNA-minicircle ATPase assays to establish that RSC, as well as its isolated ATPase subunit Sth1, are DNA translocases. RSC/Sth1 ATPase activity is stimulated by single-stranded DNA, suggesting that Sth1 tracks along one strand of the DNA duplex. Each RSC complex appears to contain a single molecule of Sth1, and isolated Sth1 is capable of nucleosome remodeling. We propose that the remodeling enzyme remains in a fixed position on the octamer and translocates a segment of DNA (with accompanying DNA twist), which breaks histone-DNA contacts and propagates as a wave of DNA around the octamer. The demonstration of DNA translocation presented here provides a mechanistic basis for this DNA wave. To test the relative contribution of twist to remodeling, we use nucleosomes containing nicks in precise locations to uncouple twist and translocation. Nucleosomes bearing nicks are remodeled less efficiently than intact nucleosomes. These results suggest that RSC and Sth1 are DNA translocases that use both DNA translocation and twist to remodel nucleosomes efficiently.

Structural and Thermodynamic Characterization of the DNA Binding Properties of a Triple Alanine Mutant of MATα2

Triply mutated MATα2 protein, α2-3A, in which all three major groove-contacting residues are mutated to alanine, is defective in binding DNA alone or in complex with Mcm1 yet binds with MATa1 with near wild-type affinity and specificity. To gain insight into this unexpected behavior, we determined the crystal structure of the a1/α2-3A/DNA complex. The structure shows that the triple mutation causes a collapse of the α2-3A/DNA interface that results in a reorganized set of α2-3A/DNA contacts, thereby enabling the mutant protein to recognize the wild-type DNA sequence. Isothermal titration calorimetry measurements reveal that a much more favorable entropic component stabilizes the a1/α2-3A/DNA complex than the α2-3A/DNA complex. The combined structural and thermodynamic studies provide an explanation of how partner proteins influence the sequence specificity of a DNA binding protein.

The X-ray Crystal Structure of the NF-κB p50·p65 Heterodimer Bound to the Interferon β-κB Site

We have determined the x-ray crystal structure of the transcription factor NF-kappaB p50.p65 heterodimer complexed to kappaB DNA from the cytokine interferon beta enhancer (IFNbeta-kappaB). To better understand how the binding modes of NF-kappaB on its two best studied DNA targets might contribute to promoter-specific transcription, this structure is compared with the previously determined complex crystal structure containing NF-kappaB bound to the Ig kappa light chain gene enhancer as well as to a second NF-kappaB.Ig kappa light chain gene enhancer complex also reported in this paper. The global binding modes of all NF-kappaB.DNA complex structures are similar, although crystal-packing interactions lead to differences between identical complexes of the same crystallographic asymmetric unit. An extensive network of stacked amino acid side chains that contribute to base-specific DNA contacts is conserved among the three complexes. Consistent with earlier reports, however, the IFNbeta-kappaB DNA is bent significantly less by NF-kappaB than is the Ig kappa light chain gene enhancer. This and other small structural changes may play a role in explaining why NF-kappaB-directed transcription is sensitive to the context of specific promoters. The precise molecular mechanism behind the involvement of the high mobility group protein I(Y) in interferon beta enhanceosome formation remains elusive.

Paper Alert

Paper Alert

Characterizing the DNA Contacts and Cooperative Binding of F Plasmid TraM to Its Cognate Sites at oriT

TraM is a DNA binding protein required for conjugative transfer of the self-transmissible IncF group of plasmids, including F, R1, and R100. F TraM binds to three sites in F oriT: two high affinity binding sites, sbmA and sbmB, which are direct repeats of nearly identical sequence involved in the autoregulation of the traM gene; and a lower affinity site, sbmC, an inverted repeat important for transfer, which is situated nearest to the nic site where transfer originates. TraM bound cooperatively to its binding sites at oriT; the presence of sbmA and sbmB increased the affinity for sbmC 10-fold. Bending of oriT DNA by TraM was minimal, suggesting that TraM, a tetramer, was able to loop the DNA when bound to sbmA and sbmB simultaneously. Hydroxyl radical footprinting of DNA of sbmA and sbmC revealed that TraM contacted the DNA within a region previously delineated by DNase I footprinting. TraM protected the CT bases within the sequence CTAG, which occurred at 12-base intervals on the top and bottom strand of sbmA, most consistently with other protected bases. The footprint on sbmC revealed that the predicted inverted repeats were protected by TraM with a pattern that began at the center of the repeats and radiated outward at 11-12 base intervals toward the 5'-ends of either strand. At high protein concentrations, this pattern extended beyond the footprint defined by DNase I, suggesting that the DNA was wrapped around the protein forming a nucleosome-like structure, which could aid in preparing the DNA for transfer.

The effects of estrogen-responsive element- and ligand-induced structural changes on the recruitment of cofactors and transcriptional responses by ER alpha and ER beta.

Estrogen signaling is mediated by ER alpha and -beta. ERs are converted from an inactive form to a transcriptionally active state through conformational changes induced by ligand and estrogen-responsive element (ERE) sequences. We show here that ER alpha and ER beta bind to an ERE independently from ER ligands. We found that although the binding affinity of ER beta for an ERE is 2-fold lower than that of ER alpha, both ERs use the same nucleotides for DNA contacts. We show that both EREs and ligands are independent modulators of ER conformation. Specifically, the ERE primarily determines the receptor-DNA affinity, whereas the structure of the ER ligand dictates the affinity of ER for particular cofactors. We found that the ligand-dependent cofactor transcriptional intermediary factor-2, through a distinct surface, also interacts with ER alpha preferentially and independently of ligand. The extent of interaction, however, is dependent upon the ER-ERE affinity. In transfected cells, ER alpha is more transcriptionally active than ER beta. The ERE sequence, however, determines the potency of gene induction when either ER subtype binds to an agonist. Antagonists prevent ERs from inducing transcription independently from ERE sequences. Thus, ERE- and ligand-induced structural changes are independent determinants for the recruitment of cofactors and transcriptional responses. The ability of ER alpha to differentially recruit a cofactor could contribute to ER subtype-specific gene responses.

P53 gene mutation and genetic instability in superficial multifocal esophageal squamous cell carcinoma

Tumor multicentricity is occasionally observed in esophageal squamous cell carcinoma (SCC). We studied five surgically resected superficial multifocal esophageal SCCs for p53 gene mutation and genetic instability, using DNA extracted from microdissected areas. A total of 38 target areas (TAs) were analyzed in SCC, dysplasia, basal cell hyperplasia (BCH) and normal squamous epithelium. Analysis of the replication error (RER) at 10 microsatellite loci showed microsatellite instability in all TAs, as well as in normal squamous epithelium. p53 gene mutation was identified in 28.9% (11/38 TAs). All cases showed a common missense mutation in exon 8 at codon 273 (CGT-->CAT, Arg-->His), which was DNA contact mutation in the S10 beta strand. In association with microsatellite alterations, 7 of 9 TAs with p53 mutation in exon 8 at codon 273 also showed loss of heterozygosity (LOH) of p53 gene. LOH of p53 gene was detected in 83.8% (31/37 TAs). LOH at D2S123 on 2p16 near MSH2 gene and at D3S1611 on 3p22 near MLH1 gene was detected in 65.4% (17/26) and 71.4% (10/14) TAs, respectively. Frequencies of LOH at p53 and D2S123 were similar in non-cancerous areas and SCCs. LOH of p53 and D2S123 were found in 50% (5/10 TAs) of non-cancerous areas and 60% (9/15 TAs) of SCCs. Our results suggest that genetic instability induces esophageal tumor multicentricity, and that p53 gene contact mutation together with LOH are early events of the multistage carcinogenesis of multifocal primary esophageal SCC.

Hydrophobic Residue Contributions to Sequence-Specific DNA Binding by the Bovine Papillomavirus Helicase E1

Previously, mutational analyses of the DNA binding domain of the bovine papillomavirus E1 protein (E1DBD) identified several hydrophobic residues that are critical for DNA binding activity (M. West, D. Flanery, K. Woytek, D. Rangasamy, and V. G. Wilson, 2001, J. Virol. 75, 11948–11960). Hydrophobic interactions of nonpolar amino acid side chains can contribute to the function of DNA binding proteins through both conformational effects and direct interaction with nucleotides. To further investigate the role of hydrophobic residues in E1DBD function, a more extensive site-directed mutational analysis of hydrophobic amino acids was conducted. Alanine substitutions were made at residues V196, F197, F217, F, 237, V246, L249, and F276, and the mutants were tested for DNA binding activity in vitro and in vivo. The E1 F237A and F276A mutants were completely defective for site-specific DNA binding, while the other mutants retained partial to full wild-type binding activity. Consistent with their DNA binding defect, the F237A and F276A mutants were severely impaired for the ability to support transient in vivo replication of an origin plasmid. Combined with our previous study, five critical hydrophobic residues have been identified: F175, V193, F237, V246, and F276. These five residues localize to two internal clusters in the E1DBD structure designated hydrophobic clusters A (HCA; includes F175, V193, and F276) and B (HCB; includes F237 and V246). Amino acid side chains from residues in HCA and HCB have little surface accessibility and it is unlikely that they are involved in direct contact with DNA. HCA is distal to the DNA binding surface and presumably contributes to global conformational organization of the E1DBD. HCB is positioned beneath the DNA contact surface and we propose that it serves as an anchor or platform device to stabilize the DNA-binding element. A comparable hydrophobic cluster is present in the corresponding position in the T antigen DBD and likely serves a similar function.