Information analysis of Fis binding sites

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.     

An analysis of the relationship between hydration and protein-DNA interactions

Eleven protein-DNA crystal structures were analyzed to test the hypothesis that hydration sites predicted in the first hydration shell of DNA mark the positions where protein residues hydrogen-bond to DNA. For nine of those structures, protein atoms, which form hydrogen bonds to DNA bases, were found within 1.5 A of the predicted hydration positions in 86% of the interactions. The correspondence of the predicted hydration sites with the hydrogen-bonded protein side chains was significantly higher for bases inside the conserved DNA recognition sequences than outside those regions. In two CAP-DNA complexes, predicted base hydration sites correctly marked 71% (within 1.5 A) of protein atoms, which form hydrogen bonds to DNA bases. Phosphate hydration was compared to actual protein binding sites in one CAP-DNA complex with 78% marked contacts within 2.0 A. These data suggest that hydration sites mark the binding sites at protein-DNA interfaces.     

Structure-activity studies of the regulatory interaction of the 10 kilodalton C-terminal fragment of caldesmon with actin and the effect of mutation of caldesmon residues 691-696

We have used isotope-edited nuclear magnetic resonance spectroscopy, binding studies, and ATPase activity assays to investigate the interaction with F-actin of the 10 kDa C-terminal 658C fragment of chicken gizzard caldesmon and two site-directed mutants of this fragment. Simultaneous dual-sited contacts with F-actin are observed for the segments of the 658C sequence flanking tryptophan residues 692 and 722. Competition experiments showed that both 658C contacts with actin are displaced by substoichiometric concentrations of the short inhibitory region of troponin-I indicative of different binding sites on actin for these regions of troponin-I and caldesmon. Substitution of caldesmon serine-702 by aspartic acid within the spacer region linking the two actin contacts of 658C led to weaker binding but with retention of equivalent affinity for each interaction site. Differential binding affinity of the two sites was achieved by replacement of the sequence Glu691-Trp-Leu-Thr-Lys-Thr696 by Pro-Gly-His-Tyr-Asn-Asn. Consistent with these data, the concentration of this Cg1 mutant required to achieve 50% inhibition of actin-tropomyosin-activated myosin ATPase was 4-fold greater than found for the 658C fragment. Although calmodulin binding to Cg1 was observed, calmodulin proved ineffective in relieving the inhibition induced by the binding of this mutant to actin. These results are discussed in light of the actin contacts which are involved in the inhibitory activity possessed by different regions of the C-terminus of caldesmon.     

DNA bending is essential for the site-specific recognition of DNA response elements by the DNA binding domain of the tumor suppressor protein p53

We have used circular permutation assays to determine the extent and location of the DNA bend induced by the DNA binding domain of human wild type p53 (p53DBD) upon binding to several naturally occurring DNA response elements. We have found that p53DBD binding induces axial bending in all of the response elements investigated. In particular, response elements having a d(CATG) sequence at the junction of two consensus pentamers in each half-site favor highly bent complexes (bending angle is approximately 50 degrees ), whereas response elements having d(CTTG) bases at this position are less bent (bending angles from approximately 37 to approximately 25 degrees ). Quantitative electrophoretic mobility shift assays of different complexes show a direct correlation between the DNA bending angle and the binding affinity of the p53DBD with the response elements, i.e. the greater the stability of the complex, the more the DNA is bent by p53DBD binding. The study provides evidence that the energetics of DNA bending, as determined by the presence or absence of flexible sites in the response elements, may contribute significantly to the overall binding affinity of the p53DBD for different sequences. The results therefore suggest that both the structure and the stability of the p53-DNA complex may vary with different response elements. This variability may be correlated with variability in p53 function.     

The transactivation region of the fis protein that controls site-specific DNA inversion contains extended mobile beta-hairpin arms

The Fis protein regulates site-specific DNA inversion catalyzed by a family of DNA invertases when bound to a cis-acting recombinational enhancer. As is often found for transactivation domains, previous crystal structures have failed to resolve the conformation of the N-terminal inversion activation region within the Fis dimer. A new crystal form of a mutant Fis protein now reveals that the activation region contains two beta-hairpin arms that protrude over 20 A from the protein core. Saturation mutagenesis identified the regulatory and structurally important amino acids. The most critical activating residues are located near the tips of the beta-arms. Disulfide cross-linking between the beta-arms demonstrated that they are highly flexible in solution and that efficient inversion activation can occur when the beta-arms are covalently linked together. The emerging picture for this regulatory motif is that contacts with the recombinase at the tip of the mobile beta-arms activate the DNA invertase in the context of an invertasome complex.     

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.     

In vivo identification of intermediate stages of the DNA inversion reaction catalyzed by the Salmonella Hin recombinase

The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.     

Effects of FR173657, a non-peptide B2 antagonist, on kinin-induced hypotension, visceral and peripheral oedema formation and bronchoconstriction

The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.     

Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.     

DNA complexes obtained with the integron integrase IntI1 at the attI1 site

Integrons are genetic elements that are able to capture genes by a site-specific recombination mechanism. Integrons contain a gene coding for a lambda-like integrase that carries out site-specific recombination by interacting with two different target sites; the attI site and the palindromic sequence attC (59 base element). Cassette integrations usually involve the attI site, while cassette excisions use attC . Therefore, the integrase should bind both sites to cleave DNA and perform site-specific recombination reactions. We have used purified maltose-binding protein fused with the integrase (MBP-IntI1) and native IntI1 protein and gel retardation assays with fragments containing the complete and partial attI1 site to show formation of four complexes in this region. Chemical modification of specific nucleotides within the attI1 site was used to investigate their interference with binding of the integrase protein. We attribute IntI1 specific binding to four regions in the attI1 site and a GTTA consensus sequence is found in three of the four regions. Interference by modified guanine and thymine residues in the DNA major groove and adenine residues in the minor groove were observed, indicating that the integrase interacts with both sides of the helix. Binding of IntI1 to attC is also discussed.     

Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element-binding factor (ERF domain) in plant

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pM range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity.     

Rapid sequence evolution of the mammalian sex-determining gene SRY

In mammals, induction of male sex determination requires the Y-chromosome gene SRY. SRY encodes a protein with a central 'high mobility group' domain (HMG box) of about 78 amino acids. HMG boxes are found in a wide variety of proteins that bind to DNA with high affinity but differing degrees of sequence specificity. The human SRY protein binds to linear DNA with sequence specificity and to cruciform DNA structures without sequence specificity. The DNA-binding activity of the SRY protein resides in the HMG box and mutations in this region are associated with sex reversal in XY females. No function has been ascribed to the portions of the SRY protein outside the HMG box. SRY belongs to a family of genes that are related by sequence homology within the DNA-binding domain: the genes most similar to SRY (> 60%) have been named SOX genes (SRY box genes). None of the known SOX genes is homologous to SRY outside the HMG-box region. Although SRY is an important developmental regulator, its sequence is poorly conserved between species apart from the HMG-box domain. Here we investigate the coding sequence of SRY in primates and find that evolution has been rapid in the regions flanking the conserved domain. The high degree of sequence divergence and the frequency of non-synonymous mutations suggest either that the majority of the coding sequence has no functional significance or that directional selection has occurred.     

The cytoskeletal protein talin contains at least two distinct vinculin binding domains

We have mapped the vinculin-binding sites in the cytoskeletal protein talin as well as those sequences which target the talin molecule to focal contacts. Using a series of overlapping talin-fusion proteins expressed in E. coli and 125I-vinculin in both gel-overlay and microtitre well binding assays, we present evidence for three separable binding sites for vinculin. All three are in the tail segment of talin (residues 434-2541) and are recognized by the same fragment of vinculin (residues 1-258). Two sites are adjacent to each other and span residues 498-950, and the third site is more than 700 residues distant in the primary sequence. Scatchard analysis of 125I-vinculin binding to talin also indicates three sites, each with a similar affinity (Kd = 2-6 x 10(-7) M). We also detect a substoichiometric interaction of higher affinity (Kd = 3 x 10(-8) M) which remains unexplained. By expressing regions of the chicken talin molecule in heterologous cells, we have shown that the sequences required to target talin to focal contacts overlap those which bind vinculin.