Inhibition of DNA binding by human estrogen-related receptor 2 and estrogen receptor alpha with minor groove binding polyamides

Human estrogen-related receptor 2 (hERR2, ESRRB, ERRbeta, NR3B2) belongs to a class of nuclear receptors that bind DNA through sequence-specific interactions with a 5'-AGGTCA-3' estrogen response element (ERE) half-site in the major groove and an upstream 5'-TNA-3' site in the minor groove. This minor groove interaction is mediated by a C-terminal extension (CTE) of the DNA binding domain and is unique to the estrogen-related receptors. We have used synthetic pyrrole-imidazole polyamides, which bind specific sequences in the minor groove, to demonstrate that DNA binding by hERR2 is sensitive to the presence of polyamides in both the upstream minor groove CTE site and the minor groove of the ERE half-site. Thus, polyamides can inhibit hERR2 by two mechanisms, by direct steric blockage of minor groove DNA contacts mediated by the CTE and by changing the helical geometry of DNA such that major groove interactions are weakened. To confirm the generality of the latter approach, we show that the dimeric human estrogen receptor alpha (hERalpha, ESR1, NR3A1), which binds in the major groove of the ERE, can be inhibited by a polyamide bound in the opposing minor groove of the ERE. These results highlight two mechanisms for inhibition of protein-DNA interactions and extend the repertoire of DNA recognition motifs that can be inhibited by polyamides. These molecules may thus be useful for controlling expression of hERR2- or hERalpha-responsive genes.     

Mechanism of damage recognition by Escherichia coli DNA photolyase

Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.     

The thermodynamics of polyamide-DNA recognition: hairpin polyamide binding in the minor groove of duplex DNA

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.     

Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts

Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia, and we have shown that it is a structure-specific binding protein. Here we examine the molecular interactions that contribute to recognition of the glycosylated base in synthetic DNA substrates using modification interference, modification protection, DNA footprinting, and photocross-linking techniques. We find that the two primary requirements for J-DNA recognition include contacts at base J and a base immediately 5' of J (J-1). Methylation interference analysis indicates that the requirement of the base at position J-1 is due to a major groove contact independent of the sequence. DNA footprinting of the JBP.J-DNA complex with 1,10-phenanthroline-copper demonstrates that JBP contacts the minor groove at base J. Substitution of the thymine moiety of J with cytosine reduces the affinity for JBP approximately 15-fold. These data indicate that the sole sequence dependence for JBP binding may lie in the thymine moiety of base J and that recognition requires only two specific base contacts, base J and J-1, within both the major and minor groove of the J-DNA duplex.     

1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. 1H NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.     

Expanding the recognition of the minor groove of DNA by incorporation of beta-alanine in hairpin polyamides

In order to expand the recognition code by hairpin polyamides to include DNA sequences of the type 5'-CWWC-3' two polyamides, PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) and PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) were synthesized which have in common an Py/Im pair in the terminal position for targeting C x G but differ with respect to internal placement of a beta-alanine residue. The equilibrium association constants (Ka) were determined at four DNA sites which differ at a single common position, 5'-TNTACA-3' (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that the eight-ring hairpin PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) binds the four binding sites with similar affinities, Ka = 1.3-1.9 x 10(10) M(-1) indicating that there is no preference for the position N. In contrast, a redesigned polyamide PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) that places an internal flexible aliphatic beta-alanine to the 5'-side of a key imidazole group bound the match site 5'-TCTACA-3' with high affinity and good sequence discrimination (Ka(match) = 4.9 x 10(10) M(-1) and the single base pair mismatch sites with 5- to 25-fold lower affinity). These results expand the repertoire of sequences targetable by hairpins and emphasize the importance of beta-alanine as a key element for minor groove recognition.     

Structural effects of DNA sequence on T.A recognition by hydroxypyrrole/pyrrole pairs in the minor groove

Synthetic polyamides composed of three types of aromatic amino acids, N-methylimidazole (Im), N-methylpyrrole (Py) and N-methyl-3-hydroxypyrrole (Hp) bind specific DNA sequences as antiparallel dimers in the minor groove. The side-by-side pairings of aromatic rings in the dimer afford a general recognition code that allows all four base-pairs to be distinguished. To examine the structural consequences of changing the DNA sequence context on T.A recognition by Hp/Py pairs in the minor groove, crystal structures of polyamide dimers (ImPyHpPy)(2) and the pyrrole counterpart (ImPyPyPy)(2) bound to the six base-pair target site 5'-AGATCT-3' in a ten base-pair oligonucleotide have been determined to a resolution of 2.27 and 2.15 A, respectively. The structures demonstrate that the principles of Hp/Py recognition of T.A are consistent between different sequence contexts. However, a general structural explanation for the non-additive reduction in binding affinity due to introduction of the hydroxyl group is less clear. Comparison with other polyamide-DNA cocrystal structures reveals structural themes and differences that may relate to sequence preference.     

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.     

Structure of homeodomain-leucine zipper/DNA complexes studied using hydroxyl radical cleavage of DNA and methylation interference

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3' end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence.     

Crystal structure of an engrailed homeodomain-DNA complex at 2.8 A resolution: a framework for understanding homeodomain-DNA interactions

The crystal structure of a complex containing the engrailed homeodomain and a duplex DNA site has been determined at 2.8 A resolution and refined to a crystallographic R factor of 24.4%. In this complex, two separate regions of the 61 amino acid polypeptide contact a TAAT subsite. An N-terminal arm fits into the minor groove, and the side chains of Arg-3 and Arg-5 make contacts near the 5' end of this "core consensus" binding site. An alpha helix fits into the major groove, and the side chains of IIe-47 and Asn-51 contact base pairs near the 3' end of the TAAT site. This "recognition helix" is part of a structurally conserved helix-turn-helix unit, but these helices are longer than the corresponding helices in the lambda repressor, and the relationship between the helix-turn-helix unit and the DNA is significantly different.     

Dual DNA recognition codes of a short peptide derived from the basic leucine zipper protein EmBP1

Sequence-specific DNA binding of short peptide dimers derived from a plant basic leucine zipper protein EmBP1 was studied. A homodimer of the EmBP1 basic region peptide recognized a palindromic DNA sequence, and a heterodimer of EmBP1 and GCN4 basic region peptides targets a non-palindromic DNA sequence when a beta-cyclodextrin/adamantane complex is utilized as a dimerization domain. A homodimer of the EmBP1 basic region peptide binds the native EmBP1 binding 5'-GCCACGTGGC-3' and the native GCN4 binding 5'-ATGACGTCAT-3' sequences with almost equal affinity in the alpha-helical conformation, indicating that the basic region of EmBP1 by itself has a dual recognition codes for the DNA sequences. The GCN4 basic region peptide binds 5'-ATGAC-3' in the alpha-helical conformation, but it neither shows affinity nor helix formation with 5'-GCCAC-3'. Because native EmBP1 forms 100 times more stable complex with 5'-GCCACGTGGC-3' over 5'-ATGACGTCAT-3', our results suggest that the sequence-selectivity of native EmBP1 is dictated by the structure of leucine zipper dimerization domain including the hinge region spanning between the basic region and the leucine zipper.     

The nonconserved hinge region and distinct amino-terminal domains of the ROR alpha orphan nuclear receptor isoforms are required for proper DNA bending and ROR alpha-DNA interactions.

ROR alpha 1 and ROR alpha 2 are two isoforms of a novel member of the steroid-thyroid-retinoid receptor superfamily and are considered orphan receptors since their cognate ligand has yet to be identified. These putative receptors have previously been shown to bind as monomers to a DNA recognition sequence composed of two distinct moieties, a 3' nuclear receptor core half-site AGGTCA preceded by a 5' AT-rich sequence. Recognition of this bipartite hormone response element (RORE) requires both the zinc-binding motifs and a group of amino acid residues located at the carboxy-terminal end of the DNA-binding domain (DBD) which is referred to here as the carboxy-terminal extension. In this report, we show that binding of ROR alpha 1 and ROR alpha 2 to the RORE induces a large DNA bend of approximately 130 degrees which may be important for receptor function. The overall direction of the DNA bend is towards the major groove at the center of the 3' AGGTCA half-site. The presence of the nonconserved hinge region which is located between the DBD and the putative ligand-binding domain (LBD) or ROR alpha is required for maximal DNA bending. Deletion of a large portion of the amino-terminal domain (NTD) of the ROR alpha protein does not alter the DNA bend angle but shifts the DNA bend center 5' relative to the bend induced by intact ROR alpha. Methylation interference studies using the NTD-deleted ROR alpha 1 mutant indicate that some DNA contacts in the 5' AT-rich half of the RORE are also shifted 5', while those in the 3' AGGTCA half-site are unaffected. These results are consistent with a model in which the ROR alpha NTD and the nonconserved hinge region orient the zinc-binding motifs and the carboxy-terminal extension of the ROR alpha DBD relative to each other to achieve proper interactions with the two halves of its recognition site. Transactivation studies suggest that both protein-induced DNA bending and protein-protein interactions are important for receptor function.     

The recognition domain of the BpuJI restriction endonuclease in complex with cognate DNA at 1.3-A resolution

Type IIS restriction endonucleases recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. The restriction endonuclease BpuJI recognizes the asymmetric sequence 5′-CCCGT; however, it cuts at multiple sites in the vicinity of the target sequence. BpuJI consists of two physically separate domains, with catalytic and dimerization functions in the C-terminal domain and DNA recognition functions in the N-terminal domain. Here we report the crystal structure of the BpuJI recognition domain bound to cognate DNA at 1.3-Å resolution. This region folds into two winged-helix subdomains, D1 and D2, interspaced by the DL subdomain. The D1 and D2 subdomains of BpuJI share structural similarity with the similar subdomains of the FokI DNA-binding domain; however, their orientations in protein-DNA complexes are different. Recognition of the 5′-CCCGT target sequence is achieved by BpuJI through the major groove contacts of amino acid residues located on both the helix-turn-helix motifs and the N-terminal arm. The role of these interactions in DNA recognition is also corroborated by mutational analysis.