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The process of DNA recognition by the activated form of the restriction endonuclease from E. coli K involves three enzyme-DNA complexes which can be differentiated experimentally. These are: an initial complex formed at a nonspecific site; a recognition complex involving the host specificity site; and a cleavage complex dependent on the presence of ATP.
Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.
The products of the proto-oncogenes c-fos and c-jun form a tight protein complex that is a major component of the transcription factor AP1. To analyze the role of fos in the binding of this complex to the AP1 DNA recognition sequence and the mechanism of interaction in further detail, we have expressed a fos protein in E. coli using an expression vector containing the temperature-inducible lambda PL promoter and a synthetic translational start codon. The fos protein encoded by this construct (termed Baf) was enriched by biochemical purification techniques and was found to form a specific complex with c-jun obtained by in vitro transcription/translation. As shown in gel retardation assays, the baf/jun complex binds to the AP1 DNA recognition sequence with high affinity, while no significant binding was observed with either of the individual protein components, indicating cooperative DNA binding of the two proteins. The fact that the bacterial baf protein does not undergo glycosylation indicates that the post-translational modification of eukaryotic c-fos with N-acetylglucosamine is not required for the formation of a stable fos/jun/DNA complex.
To elucidate the basis of sequence-specific single-stranded (ss) DNA recognition by K homology (KH) domains, we have solved the solution structure of a complex between the KH3 domain of the transcriptional regulator heterogeneous nuclear ribonucleoprotein K (hnRNP K) and a 10mer ssDNA. We show that hnRNP K KH3 specifically recognizes a tetrad of sequence 5'd-TCCC. The complex is stabilized by a dense network of methyl-oxygen hydrogen bonds involving the methyl groups of three isoleucine residues and the O2 and N3 atoms of the two central cytosine bases. Comparison with the recently solved structure of a specific protein-ssDNA complex involving the KH3 and KH4 domains of the far upstream element (FUSE) binding protein FBP suggests that the amino acid located five residues N-terminal of the invariant GXXG motif, which is characteristic of all KH domains, plays a crucial role in discrimination of the first two bases of the tetrad.
The three-dimensional structure of an HNF-3/fork head DNA-recognition motif complexed with DNA has been determined by X-ray crystallography at 2.5 A resolution. This alpha/beta protein binds B-DNA as a monomer, through interactions with the DNA backbone and through both direct and water-mediated major and minor groove base contacts, inducing a 13 degrees bend. The transcription factor fold is very similar to the structure of histone H5. In its amino-terminal half, three alpha-helices adopt a compact structure that presents the third helix to the major groove. The remainder of the protein includes a twisted, antiparallel beta-structure and random coil that interacts with the minor groove.
ZFY, a male-associated Zn-finger protein encoded by the human Y chromosome, exhibits a distinctive two-finger repeat: whereas odd-numbered domains fit a general consensus, even-numbered domains exhibit systematic differences. Do these odd and even sequences encode structurally distinct surfaces for DNA recognition? As a first step toward answering this question, we have recently described the sequential 1H NMR assignment of a representative nonconsensus Zn finger (designated ZFY-6T) based on 2D NMR studies of a 30-residue peptide [Kochoyan, M., Havel, T.F., Nguyen, D.T., Dahl, C.E., Keutmann, H. T., & Weiss, M.A. (1991) Biochemistry 30, 3371-3386]. Initial structural modeling by distance geometry/simulated annealing (DG/SA) demonstrated that this peptide retained the N-terminal beta-hairpin and C-terminal alpha-helix (beta beta alpha motif) observed in consensus Zn fingers. However, the precision of this initial structure was limited by resonance overlap, which led to ambiguities in the assignment of key NOEs in the hydrophobic core. In this paper these ambiguities are resolved by selective deuterium labeling, enabling a refined structure to be calculated by DG/SA and restrained molecular dynamics. These calculations provide a detailed view of the hydrophobic core and protein surface, which are analyzed in reference to previously characterized Zn fingers. Variant (even) and consensus (odd) aromatic residues Y10 and F12, shown in an "aromatic swap" analogue to provide equivalent contributions to the hydrophobic core [Weiss, M.A., & Keutmann, H.T. (1990) Biochemistry 29, 9808-9813], nevertheless exhibit striking differences in packing interactions: Y10--but not F12--contributes to a contiguous region of the protein surface defined by putative specificity-determining residues. Alternating surface architectures may have implications for the mechanism of DNA recognition by the ZFY two-finger repeat.
Duplex DNA recognition by oligonucleotide-directed triple helix formation is generally limited to homopurine target domains. Various approaches have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopurine domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs between domains. Using assays of electrophoretic mobility and chemical protection, we have explored in detail whether such strategies are of benefit in designing high-affinity probes for a natural DNA sequence in the human p53 gene. This target site contains three adjacent, purine-rich domains on opposite DNA strands. Our results show that (i) a modest but statistically significant enhancement in affinity can be achieved for this sequence by designing an oligonucleotide that simultaneously recognizes all three purine domains, (ii) correction of a pyrimidine interruption in one purine domain does not dramatically alter this result, (iii) the relative energetic and structural contributions attributable to recognition of each purine domain can be assessed using probes with combinations of specific and nonspecific nucleotide sequences, and (iv) probe affinity is not correlated with the apparent number of base triplets for certain complexes. These data suggest that unfavorable free energy changes may be associated with alternation between triple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enhancement was not observed using alternate strand DNA recognition at this natural target sequence. We therefore conclude that such enhancement is sequence dependent.
The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.
The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.
Restriction endonucleases were tested for their ability to catalyze the cleavage of mismatch-containing recognition sites in DNA. These mismatched base pairs were T.G, U.G, or A.C in covalently closed, circular heteroduplexes prepared by in vitro extension of chemically synthesized oligonucleotide primers annealed to a bacteriophage M13-derived viral DNA. None of the restriction enzymes was able to completely cleave the mismatch-containing recognition sites under standard conditions. However, three of them, SmaI, SalI, and SstI, catalyzed partial digestion leading to an accumulation of DNA singly nicked at the mismatched recognition site. The ability of SmaI and SstI to partially cleave at a mismatch was shown to depend on the nature and position of the mismatch within the corresponding recognition site. In contrast, little or no digestion was obtained with AccI, HincII, HindIII, and KpnI at mismatch-containing sites. Therefore, in some cases a transition-type substitution in only one strand of a recognition site inhibits restriction endonuclease-catalyzed digestion at that site although in others partial digestion occurs.
A comprehensive analysis of interfacial water molecules in the structures of 109 unique protein-DNA complexes is presented together with a new view on their role in protein-DNA recognition. Location of interfacial water molecules as reported in the crystal structures and as emerging from a series of molecular dynamics studies on protein-DNA complexes with explicit solvent and counterions, was analyzed based on their acceptor, donor hydrogen bond relationships with the atoms and residues of the macromolecules, electrostatic field calculations and packing density considerations. Water molecules for the purpose of this study have been categorized into four classes: viz. (I) those that contact both the protein and the DNA simultaneously and thus mediate recognition directly; (II) those that contact either the protein or the DNA exclusively via hydrogen bonds solvating each solute separately; (III) those that contact the hydrophobic groups in either the protein or the DNA; and, lastly (IV) those that contact another water molecule. Of the 17,963 crystallographic water molecules under examination, about 6% belong to class I and 76% belong to class II. About three-fourths of class I and class II water molecules are exclusively associated with hydrogen bond acceptor atoms of both protein and DNA. Noting that DNA is polyanionic, it is significant that a majority of the crystallographically observed water molecules as well as those from molecular dynamics simulations should be involved in facilitating binding by screening unfavorable electrostatics. Less than 2% of the reported water molecules occur between hydrogen bond donor atoms of protein and acceptor atoms of DNA. These represent cases where protein atoms cannot reach out to DNA to make favorable hydrogen bond interactions due to packing/structural restrictions and interfacial water molecules provide an extension to side-chains to accomplish hydrogen bonding.
Anti-double-stranded DNA monoclonal antibodies against a viral transcriptional regulatory site are capable of discriminating single-base replacements with affinities of 1 x 10(-)(9) M, which were optimized for the length of the duplex used as the immunogen. Their affinity for DNA duplexes of increasing length is lower, but reaches a plateau at 2 x 10(-)(8) M, still a fairly high affinity compared to those of most known natural anti-DNA antibodies. The ability of the antibodies to bind to a 166 bp DNA fragment containing the specific sequence strongly suggests that these have the potential of binding the specific sequence within larger genomic DNA fragments. Electrostatic interactions do not play a significant role, the opposite of what is observed in natural DNA binding interfaces. In addition, the insensitivity of the antibody-DNA interaction to solute effects is indicative of a marginal participation of water molecules at the interface compared to the level of participation at the natural E2-DNA interface. Spectroscopic evidence of base unstacking strongly suggests substantial denaturation of antibody-bound DNA, in agreement with thermodynamic results that show an unusual positive heat capacity change, which could be explained at least in part by the exposure of DNA bases upon binding. Lower local DNA stability cooperates with sequence recognition in producing the highest binding affinity. A slow rate of antibody-DNA association indicates an energy barrier imposed by conformational rearrangements, as opposed to an electrostatically assisted diffusion-controlled collision in the E2 DNA binding domain. While the E2-DNA interaction takes place through a typical direct readout mechanism, the anti-double-stranded DNA monoclonal antibody-DNA interaction could be viewed as a distinctive case of indirect readout with a significant distortion in the DNA conformation. However, the precise mechanism with which the DNA bases are accommodated in the antibody combining site will require structural analysis at atomic resolution. These results constitute a first stage for unveiling the unusual molecular recognition mechanism of a specific DNA sequence by antibodies. This mechanism could represent the strategy with which the immune system tightly and specifically recognizes a DNA antigen.
The molecular basis for the DNA binding specificity of the thyroid transcription factor 1 homeodomain (TTF-1HD) has been investigated. Methylation and ethylation interference experiments show that the TTF-1HD alone recapitulates the DNA binding properties of the entire protein. Studies carried out with mutant derivatives of TTF-1HD indicate a precise correspondence of some of its amino acid residues with specific bases in its binding site, allowing a crude orientation of the TTF-1HD within the protein-DNA complex. TTF-1HD shows an overall geometry of interaction with DNA similar to that previously observed for Antennapedia class HDs, even though the binding specificities of these two types of HDs are distinct. We demonstrate that the crucial difference between the binding sites of Antennapedia class and TTF-1 HDs is in the motifs 5'-TAAT-3', recognized by Antennapedia, and 5'-CAAG-3', preferentially bound by TTF-1. Furthermore, the binding of wild type and mutants TTF-1 HD to oligonucleotides containing either 5'-TAAT-3' or 5'-CAAG-3' indicate that only in the presence of the latter motif the Gln50 in TTF-1 HD is utilized for DNA recognition. Since the Gln at position 50 is an essential determinant for DNA binding specificity for several other HDs that bind to 5'-TAAT-3' containing sequences, we suggest that utilization by different HDs of key residues may depend on the sequence context and probably follows a precise hierarchy of contacts.
New six zinc finger protein DSp1 was created by connecting two Sp1 DNA binding domains. The DNA recognition characteristics of DSp1 demonstrate that (1) this multi finger protein binds eighteen contiguous base pairs of DNA sequence, (2) each zinc finger wraps around DNA major groove, and (3) two Sp1 domains have nonequivalent contribution to the DNA binding of DSp1.
Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.
Boc-protected benzimidazole-pyrrole, benzimidazole-imidazole, and benzimidazole-methoxypyrrole amino acids were synthesized and incorporated into DNA binding polyamides, comprised of N-methyl pyrrole and N-methyl imidazole amino acids, by means of solid-phase synthesis on an oxime resin. These hairpin polyamides were designed to determine the DNA recognition profile of a side-by-side benzimidazole/imidazole pair for the designated six base pair recognition sequence. Equilibrium association constants of the polyamide-DNA complexes were determined at two of the six base pair positions of the recognition sequence by quantitative DNase I footprinting titrations on DNA fragments each containing matched and single base pair mismatched binding sites. The results indicate that the benzimidazole-heterocycle building blocks can replace pyrrole-pyrrole, pyrrole-imidazole, and pyrrole-hydroxypyrrole constructs while retaining relative site specifities and subnanomolar match site affinities. The benzimidazole-containing hairpin polyamides represent a novel class of DNA binding ligands featuring tunable target recognition sequences combined with the favorable properties of the benzimidazole type DNA minor groove binders.
The high mobility group (HMG) chromosomal proteins, which are common to all eukaryotes, bind DNA in a non-sequence-specific fashion to promote chromatin function and gene regulation. They interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in V(D)J recombination and in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA. The X-ray crystal structure, at 2.2 A resolution, of the HMG domain of the Drosophila melanogaster protein, HMG-D, bound to DNA provides the first detailed view of a chromosomal HMG domain interacting with linear DNA and reveals the molecular basis of non-sequence-specific DNA recognition. Ser10 forms water-mediated hydrogen bonds to DNA bases, and Val32 with Thr33 partially intercalates the DNA. These two 'sequence-neutral' mechanisms of DNA binding substitute for base-specific hydrogen bonds made by equivalent residues of the sequence-specific HMG domain protein, lymphoid enhancer factor-1. The use of multiple intercalations and water-mediated DNA contacts may prove to be generally important mechanisms by which chromosomal proteins bind to DNA in the minor groove.
We have constructed mutations in what we predict to be the DNA-recognition helix of Klebsiella pneumoniae NtrC, which regulates transcription from promoters under global nitrogen control. Mutations which disrupt the helix lead to complete loss of function. All point mutants tested were able to activate transcription from the sigma 54-dependent glnA promoter, but only those retaining some ability to recognise NtrC binding sites, as evidenced by their ability to repress the ntrB promoter and the upstream glnA promoter, were able to activate the nifL promoter. One mutant, which contained an amino acid substitution in the turn of the DNA-binding motif as well as in the recognition helix, suppressed mutations in the NtrC binding sites upstream from the nifL promoter, but only if both sites bore equivalent transitions. This confirms that the DNA-binding motif for this class of transcriptional activator has been correctly identified and suggests that binding of NtrC can be cooperative.