Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction

A simple, direct method for the detection of DNA–protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purβ (mPurβ), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurβ and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurβ were 25 and 3 μg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.     

Introduction of hematoxylin as an electroactive label for DNA biosensors and its employment in detection of target DNA sequence and single-base mismatch in human papilloma virus corresponding to oligonucleotide

For the detection of DNA hybridization, a new electrochemical biosensor was developed on the basis of the interaction of hematoxylin with 20-mer deoxyoligonucleotides (from human papilloma virus, HPV). The study was performed based on the interaction of hematoxylin with an alkanethiol DNA probe self-assembled gold electrode (ss-DNA/AuE) and its hybridization form (ds-DNA/AuE). The optimum conditions were found for the immobilization of HPV probe on the gold electrode (AuE) surface and its hybridization with the target DNA. Electrochemical detection of the self-assembled DNA and the hybridization process were performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the accumulated hematoxylin at the modified electrode was electroactive. Observing a remarkable difference between the voltammetric signals of the hematoxylin obtained from different hybridization samples (non-complementary, mismatch and complementary DNAs), we confirmed the potential of the developed biosensor in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 12.5 nM to 350.0 nM, and the detection limit was 3.8 nM.     

DNA mismatch correction in Haemophilus influenzae: characterization of MutL, MutH and their interaction

Haemophilus influenzae DNA mismatch repair proteins, MutS, MutL and MutH, are functionally characterized in this study. Introduction of mutS, mutL and mutH genes of H. influenzae resulted in complementation of the mismatch repair activity of the respective mutant strains of Escherichia coli to varying levels. DNA binding studies using H. influenzae MutH have shown that the protein is capable of binding to any DNA sequence non-specifically in a co-operative and metal independent manner. Presence of MutL and ATP in the binding reaction resulted in the formation of a more specific complex, which indicates that MutH is conferred specificity for binding hemi-methylated DNA through structural alterations mediated by its interaction with MutL. To study the role of conserved amino acids Ile213 and Leu214 in the helix at the C-terminus of MutH, they were mutated to alanine. The mutant proteins showed considerably reduced DNA binding and nicking, as well as MutL-mediated activation. MutH failed to nick HU bound DNA whereas MboI and Sau3AI, which have the same recognition sequence as MutH, efficiently cleaved the substrate. MutS ATPase activity was found to be reduced two-fold in presence of covalently closed circular duplex containing a mismatched base pair whereas, the activity was regained upon linearization of the circular duplex. This observation possibly suggests that the MutS clamps are trapped in the closed DNA heteroduplex. These studies, therefore, serve as the basis for a detailed investigation of the structure-function relationship among the protein partners of the mismatch repair pathway of H. influenzae.     

Comprehensive study of interactions between DNA and new electroactive Schiff base ligands. Application to the detection of singly mismatched Helicobacter pylori sequences

N,N'-Bis(3,4-dihydroxybenzylidene)-1,2-diaminobenzene (3,4-DHS) and N,N'-bis(2,5-dihydroxybenzylidene)-1,2-diaminobenzene (2,5-DHS) have been used as electrochemical probes in DNA sensing. These ligands, containing ortho and para quinone functional groups, respectively, as well as planar aromatic domains, are capable of binding to double stranded DNA (ds-DNA) more efficiently than to single stranded DNA (ss-DNA). Emphasis has been placed on the elucidation of the nature of the interaction by combining spectroscopic and electrochemical techniques. From spectrophotometric titration experiments, the binding constants of 3,4-DHS and 2,5-DHS with ds-DNA were found to be (9.0+/-0.3) x 10(3) and (3.3+/-0.2) x 10(3)M(-1), respectively. These values are consistent with a binding mode dominated by interactions with the minor groove of ds-DNA. The electroactivity of the quinone moiety in 3,4-DHS bound to DNA could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a thiolated capture probe sequence from Helicobacter pylori onto gold electrodes. After hybridization with the complementary target sequence, 3,4-DHS was accumulated within the double stranded DNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range where the quinone moiety is redox active. Using this approach, complementary target sequences of H. pylori can be quantified over the range of 8.9-22.2 microM with a detection limit of 8.3+/-0.4 microM and a linear correlation coefficient of 0.989. In addition this approach is capable of detecting hybridization of complementary sequences containing a single mismatch.     

Thermodynamics of the DNA Structural Selectivity of the Pol I DNA Polymerases from Escherichia coli and Thermus aquaticus

Understanding the thermodynamics of substrate selection by DNA polymerase I is important for characterizing the balance between replication and repair for this enzyme in vivo. Due to their sequence and structural similarities, Klenow and Klentaq, the large fragments of the Pol I DNA polymerases from Escherichia coli and Thermus aquaticus, are considered functional homologs. Klentaq, however, does not have a functional proofreading site. Examination of the DNA binding thermodynamics of Klenow and Klentaq to different DNA structures: single-stranded DNA (ss-DNA), primer-template DNA (pt-DNA), and blunt-end double-stranded DNA (ds-DNA) show that the binding selectivity pattern is similar when examined across a wide range of salt concentration, but can significantly differ at any individual salt concentration. For both proteins, binding of single-stranded DNA shifts from weakest to tightest binding of the three structures as the salt concentration increases. Both Klenow and Klentaq release two to three more ions when binding to pt-DNA and ds-DNA than when binding to ss-DNA. Klenow exhibits significant differences in the ΔC_p of binding to pt-DNA versus ds-DNA, and a difference in pI for these two complexes, whereas Klentaq does not, suggesting that Klenow and Klentaq discriminate between these two structures differently. Taken together, the data suggest that the two polymerases bind ds-DNA very differently, but that both bind pt-DNA and ss-DNA similarly, despite the absence of a proofreading site in Klentaq.     

Characterization of DNA sequence-common and sequence-specific proteins binding to cis-acting sites for cleavage of the terminal a sequence of the herpes simplex virus 1 genome.

The terminal 500-base-pair alpha sequence of the herpes simplex virus 1 genome contains signals for cleavage (Pac1 and Pac2) of unit-length DNA molecules from concatemers in unique stretches of sequences designated Ub and Uc, respectively, and a cis site for cleavage designated DR1. We report that nuclear extracts from infected cells contain factors which form two DNA-virus-specific protein complexes with components of the a sequence. Purification of the factors forming the V2 complex yielded a protein with an apparent molecular weight of 82,000 binding to DNA in a non-sequence-specific manner. Addition of Mg2+ to the purified protein-DNA probe mixture resulted in exonucleolytic degradation of the DNA. The protein was identified as the virus-specific DNase with monoclonal antibody specific for the viral enzyme. The purification of the proteins forming the V4 complex yielded two proteins with molecular weights of greater than 250,000 and 140,000 corresponding to infected cell protein 1 and to an as yet unidentified protein, respectively. These proteins formed two DNA sequence-common bands with a number of DNA probes and one sequence-specific band with probes containing both Pac2 and DR1 but not with probes containing either site alone or Pac1 and DR1. Since the DNA probe containing Pac2 and DR1 inserted into viral genome or into amplicons induced specific cleavage of the DR1 sequence whereas the nonreactive probes failed to induce the cleavage, the formation of this sequence-specific DNA-protein complex is significant and may reflect a DNA-protein interaction essential for cleavage. The possible role of the proteins identified in this study for the cleavage-packaging of viral DNA into capsids is presented.     

DNA hybridization sensor based on pentacene thin film transistor

A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs.     

DNA-binding activity of tightly-bound nonhistone chromosomal proteins in chicken liver chromatin.

We have isolated a nonhistone chromosomal protein fraction from chicken liver chromatin which possesses high affinity and preferential sequence DNA binding. Residually DNA-bound nonhistone chromosomal proteins after 2.0 M NaCl extraction of bulk chromatin are isolated. Bound proteins are released by dissociation of the complexes in 5.0 M urea/3.0 M NaCl. We have investigated the in vitro DNA-binding properties of this class. In contrast to other DNA-binding NHCP whose activities have been studied, direct DNA-binding activity is observed which is not abolished under conditions of high ionic strength (to 3.0 M NaCl). Strong preference in binding fractionated homologous DNA is observed, while binding of heterologous (E. Coli) DNA is negligible. The fractionation of homologous DNA permits the isolation of DNA for which this protein class displays strong binding preference, presumably through a concentration of binding sites. The composite data suggest sequence-specific interaction between this protein class and DNA, which is not abolished by high ionic strength.     

The study of interactions between DNA and PcrA DNA helicase by using targeted molecular dynamic simulations

DNA helicases are important enzymes involved in all aspects of nucleic acid metabolism, ranging from DNA replication and repair to recombination, rescue of stalled replication and translation. DNA helicases are molecular motors. Through conformational changes caused by ATP hydrolysis and binding, they move along the template double helix, break the hydrogen bonds between the two strands and separate the template chains, so that the genetic information can be accessed. In this paper, targeted molecular dynamic simulations were performed to study the important interactions between DNA and PcrA DNA helicase, which can not be observed from the crystal structures. The key residues on PcrA DNA helicase that have strong interactions with both double stranded DNA (ds-DNA) and single stranded DNA (ss-DNA) have been identified, and it was found that such interactions mostly exist between the protein and DNA backbone, which indicates that the translocation of PcrA is independent of the DNA sequence. The simulations indicate that the ds-DNA is separated upon ATP rebinding, rather than ATP hydrolysis, which suggests that the two strokes in the mechanism have two different major roles. Firstly, in the power stroke (ATP hydrolysis), most of the translocations of the bases from one pocket to the next occur. In the relaxation stroke (ATP binding), most of the ‘work’ is being done to ‘melt’ the DNA at the separation fork. Therefore, we propose a mechanism whereby the translocation of the ss-DNA is powered by ATP hydrolysis and the separation of the ds-DNA is powered by ATP binding.     

The histone-like protein HU binds specifically to DNA recombination and repair intermediates

The heterodimeric HU protein associated with the Escherichia coli nucleoid shares some properties with histones and HMG proteins. HU binds DNA junctions and DNA containing a nick much more avidly than double-stranded (ds-) DNA. Cells lacking HU are extremely sensitive to gamma irradiation and we wondered how HU could play a role in maintaining the integrity of the bacterial chromosome. We show that HU binds with high affinity to DNA repair and recombination intermediates, including DNA invasions, DNA overhangs and DNA forks. The DNA structural motif that HU specifically recognizes in all these structures consists of a ds-DNA module joined to a second module containing either ds- or single-stranded (ss-) DNA. The two modules rotate freely relative to one another. Binding specificity results from the simultaneous interaction of HU with these two modules: HU arms bind the ds-DNA module whereas the HU body contacts the 'variable' module containing either ds- or ss-DNA. Both structural motifs are recognized by HU at least 1000-fold more avidly than duplex DNA.     

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.     

Electrochemical spectroscopic investigations on the interaction of an ytterbium complex with DNA and their analytical applications such as biosensor

Metal ion-DNA interactions are important in nature, often changing the genetic material's structure and function. A new Yb complex of YbCl₃ (tris(8-hydroxyquinoline-5-sulfonic acid) ytterbium) was synthesized and utilized as an electrochemical indicator for the detection of DNA oligonucleotide based on its interaction with Yb(QS)₃. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction of Yb(QS)₃ with ds-DNA. It was revealed that Yb(QS)₃ presented an excellent electrochemical activity on glassy carbon electrode (GCE) and could intercalate into the double helix of double-stranded DNA (ds-DNA). The binding mechanism of interaction was elucidated on glassy carbon electrode dipped in DNA solution and DNA modified carbon paste electrode by using differential pulse voltammetry and cyclic voltammetry. The binding ratio between this complex and ds-DNA was calculated to be 1:1. The extent of hybridization was evaluated on the basis of the difference between signals of Yb(QS)₃ with probe DNA before and after hybridization with complementary DNA. With this approach, this DNA could be quantified over the range from 1 × 10⁻⁸ to 1.1 × 10⁻⁷ M. The interaction mode between Yb(QS)₃ and DNA was found to be mainly intercalative interaction. These results were confirmed with fluorescence experiments.     

The sequence specificity of homeodomain-DNA interaction

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.     

The function of Asp70, Glu77 and Lys79 in the Escherichia coli MutH protein

The MutH protein, which is part of the Dam-directed mismatch repair system of Escherichia coli, introduces nicks in the unmethylated strand of a hemi-methylated DNA duplex. The latent endonuclease activity of MutH is activated by interaction with MutL, another member of the repair system. The crystal structure of MutH suggested that the active site residues include Asp70, Glu77 and Lys79, which are located at the bottom of a cleft where DNA binding probably occurs. We mutated these residues to alanines and found that the mutant proteins were unable to complement a chromosomal mutH deletion. The purified mutant proteins were able to bind to DNA with a hemi-methylated GATC sequence but had no detectable endonuclease activity with or without MutL. Although the data are consistent with the prediction of a catalytic role for Asp70, Glu77 and Lys79, it cannot be excluded that they are also involved in binding to MutL.     

Solid phase immunoenzyme analysis of the immunospecificity of DNA from calf spleen alkylated by thiophosphamide

DNA binding activity of rabbit antiserum against calf spleen DNA's modified by thiophosphamide (DNA-T) was studied by means of solid enzyme immunoassays (ELISA). The studies demonstrated the preferential binding of the immobilized DNA-T compared to immobilized single-stranded DNA (ss-DNA) and only small preference compared to native DNA. Two antisera against DNA-T were purified by affinity chromatography on a ss-DNA-CNBr agarose from antibodies to calf spleen ss-DNA. They interacted only with the immobilized DNA-T, but not with ss-DNA or native DNA. These results demonstrated that DNA modification by thiophosphamide, decreases the immunogenicity of usual nitrogen-containing DNA bases, but detected new immunogenic specificity for adducts. Detection of new immunogenic specificity in DNA's alkylated by thiophosphamide, resulted in the development of a sensitive enzyme immunoassay for the detection of these adducts in nucleic acids, in monitoring their formation, persistence and repair damages in DNA.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).