A model for the LexA repressor DNA complex

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein–DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley-Liss, Inc.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind^–) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o ^c in the umuDC and umuD'C operons and also used the o ₉₈ ^c -recA mutation. The o ₁ ^c -umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.     

DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution

The thermal properties of two forms of the Drosophila melanogaster HMG-D protein, with and without its highly basic 26 residue C-terminal tail (D100 and D74) and the thermodynamics of their non-sequence-specific interaction with linear DNA duplexes were studied using scanning and titration microcalorimetry, spectropolarimetry, fluorescence anisotropy and FRET techniques at different temperatures and salt concentrations. It was shown that the C-terminal tail of D100 is unfolded at all temperatures, whilst the state of the globular part depends on temperature in a rather complex way, being completely folded only at temperatures close to 0 degrees C and unfolding with significant heat absorption at temperatures below those of the gross denaturational changes. The association constant and thus Gibbs energy of binding for D100 is much greater than for D74 but the enthalpies of their association are similar and are large and positive, i.e. DNA binding is a completely entropy-driven process. The positive entropy of association is due to release of counterions and dehydration upon forming the protein/DNA complex. Ionic strength variation showed that electrostatic interactions play an important but not exclusive role in the DNA binding of the globular part of this non-sequence-specific protein, whilst binding of the positively charged C-terminal tail of D100 is almost completely electrostatic in origin. This interaction with the negative charges of the DNA phosphate groups significantly enhances the DNA bending. An important feature of the non-sequence-specific association of these HMG boxes with DNA is that the binding enthalpy is significantly more positive than for the sequence-specific association of the HMG box from Sox-5, despite the fact that these proteins bend the DNA duplex to a similar extent. This difference shows that the enthalpy of dehydration of apolar groups at the HMG-D/DNA interface is not fully compensated by the energy of van der Waals interactions between these groups, i.e. the packing density at the interface must be lower than for the sequence-specific Sox-5 HMG box.     

Nanobodies targeting LexA autocleavage disclose a novel suppression strategy of SOS-response pathway

1. Download : Download high-res image (197KB)
2. Download : Download full-size image

     

AtRAD5A is a DNA translocase harboring a HIRAN domain which confers binding to branched DNA structures and is required for DNA repair in vivo

DNA lesions such as crosslinks represent obstacles for the replication machinery. Nonetheless, replication can proceed via the DNA damage tolerance pathway also known as postreplicative repair pathway. SNF2 ATPase Rad5 homologs, such as RAD5A of the model plant Arabidopsis thaliana, are important for the error‐free mode of this pathway. We able to demonstrate before, that RAD5A is a key factor in the repair of DNA crosslinks in Arabidopsis. Here, we show by in vitro analysis that AtRAD5A protein is a DNA translocase able to catalyse fork regression. Interestingly, replication forks with a gap in the leading strand are processed best, in line with its suggested function. Furthermore AtRAD5A catalyses branch migration of a Holliday junction and is furthermore not impaired by the DNA binding of a model protein, which is indicative of its ability to displace other proteins. Rad5 homologs possess HIRAN (Hip116, Rad5; N‐terminal) domains. By biochemical analysis we were able to demonstrate that the HIRAN domain variant from Arabidopsis RAD5A mediates structure selective DNA binding without the necessity for a free 3′OH group as has been shown to be required for binding of HIRAN domains in a mammalian RAD5 homolog. The biological importance of the HIRAN domain in AtRAD5A is demonstrated by our result that it is required for its function in DNA crosslink repair in vivo.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

Carboxyl terminal region of the MukB protein in Escherichia coli is essential for DNA binding activity

Abstract The purified MukB protein of Escherichia coli has DNA binding activity and nucleotide binding activity. We have isolated a mutation, mukB1013 , causing a substitution of valine at position 1379 to leucine. This mutant MukB protein was defective for DNA binding, while the ATP binding activity remained unaffected. A truncated MukB protein that is short of 109 amino acids from the C-terminus failed to bind DNA.     

DNA binding of a proflavine derivative bearing a platinum hanging residue

New platinum(II) complex of 3,6-diamine-9-[6,6-bis(2-aminohethyl)-1,6-diaminohexyl]acridine, AzaPt, has been synthesised and characterised. Behaviour of AzaPt in solution (protonation and possible self-aggregation phenomena) has been investigated by spectral methods (absorbance and fluorescence) at I = 0.1 M and 25 °C, and the equilibrium parameters of binding to calf thymus DNA have been established. Two different modes of DNA binding by the complex were detected, which depend on the polymer to dye molar ratio (P/D). At relatively low P/D values the mode was interpreted as binding by the polyamine residue external to the base pairs, while at high P/D values the binding corresponds to intercalation of the proflavine residue. Such interpretation is supported by the observed salt effect on binding and the temperature variation of the binding constants, which allowed estimating the ΔH and ΔS values contributions. Spectrophotometric analysis of the long time range binding revealed that AzaPt is involved in a slow reaction, interpreted as an attack by the platinum ion on the nucleobases. The time constant for such interaction was calculated and found to be the same order of magnitude as for processes responsible for the action of anti-tumour drugs that do covalently bind to polynucleotides.     

The structural basis for substrate specificity in DNA topoisomerase IV

Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Molecular beacons for detecting DNA binding proteins: mechanism of action

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.     

Common protein architecture and binding sites in proteases utilizing a Ser/Lys dyad mechanism

Escherichia coli signal peptidase (SPase) and E. coli UmuD protease are members of an evolutionary clan of serine proteases that apparently utilize a serine-lysine catalytic dyad mechanism. Recently, the crystallographic structure of a SPase inhibitor complex was solved elucidating the catalytic residues and the substrate binding subsites. Here we show a detailed comparison of the E. coli SPase structure to the native E. coli UmuD' structure. The comparison reveals that despite a very low sequence identity these functionally diverse enzymes share the same protein fold within their catalytic core and allows by analogy for the assignment of the cleavage-site orientation and substrate binding subsites in the UmuD(D') protease. The structural alignment of SPase and UmuD' predicts important mechanistic and structural similarities and differences within these newly characterized families of serine proteases.     

DNA binding and bending by a chloroplast-encoded HU-like protein overexpressed in Escherichia coli

The Guillardia theta chloroplast hlpA gene encodes a protein resembling bacterial histone-like protein HU. This gene was cloned and overexpressed in Escherichia coli cells, and the resulting protein product, HlpA, was purified and characterized in vitro. In addition to exhibiting a general DNA-binding activity, the chloroplast HlpA protein also strongly facilitated cyclization of a short DNA fragment in the presence of T4 DNA ligase, indicating its ability to mediate very tight DNA curvatures.     

DNA deformation energetics and protein binding

The formation of protein-DNA complexes often involves deformation of the DNA double helix. We have calculated the energy necessary to produce this deformation in 71 crystallographically determined complexes, using internal coordinate energy optimization with the JUMNA program and a generalized Born continuum solvent treatment. An analysis of the data allows deformation energy to be interpreted in terms of both local and global structural changes. We find that, in the majority of complexes, roughly 60% of the deformation energy corresponds to backbone distortion. It is also found that large changes in stacking and pairing energies are often compensated for by other, longer range, stabilizing factors. Some deformations, such as base opening, can be large, but only-produce local energetic effects. In terms of backbone distortions, the angle alpha, most often involved in alphagamma transitions, makes the most significant energetic contribution. This type of transition is twice as costly as those involving beta, or coupled epsilonzeta changes. Sugar amplitude changes are also energetically significant, in contrast to changes in phase angles.     

The binding of cationic surfactants by DNA

Isotherms for the binding of dodecyltrimethylammonium (DTA⁺) and tetradecyltrimethylammonium (TTA⁺) ions by DNA in aqueous solution at 30°C are reported. The binding isotherms were determined using a potentiometric technique with cationic surfactant-selective electrodes. The DNA concentrations used are 5 × 10^?4 and 10^?3 equiv./kg, Surfactant concentrations varying from 3 × 10^?6M to the critical micelle concentration. The influence of added NaCl (0.01 M) on the binding process is studied. The binding process is shown to be highly cooperative. Applying the binding theory of Schwarz and of Satake and Yang, binding constants and cooperativity parameters can be calculated. The binding constant K is found to be 1.2kT larger for TTA⁺ than for DTA⁺ in salt-free solution, and 1.4kT larger for TTA⁺ than for DTA⁺ in 0.01 M NaCl. The cooperativity parameter u is about 1.4kT larger for TTA⁺ in salt-free solution and 1.2kT larger in 0.01 M NaCl. It is concluded that the hydrophobic part of the bound surfactant is not completely immersed in the hydrophobic DNA core, but also interacts with other surfactant molecules. This situation is compared to the case of micelle formation.