Electrochemical properties of DNA-intercalating doxorubicin and methylene blue on n-hexadecyl mercaptan-doped 5'-thiol-labeled DNA-modified gold electrodes

Interactions between DNA-intercalating molecules, methylene blue (MB) and doxorubicin (DOX), and gold surface modified by various DNA species and n-hexadecyl mercaptan (HDM) were investigated by cyclic voltammetry (CV). Hydrophilic DOX was completely blocked by the HDM film from contacting the gold electrode whereas hydrophobic MB could readily partition into the film. Unlabeled single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) underwent non-specific adsorption on gold surface but the adsorbed DNA can be partially displaced by HDM. Thiol-labeled ssDNA and dsDNA adsorbed on gold surface via both thiol-gold linkage and non-specific interactions between DNA strands and gold. The non-specific interactions could be interrupted by the addition of HDM, forming a mixed monolayer containing both HDM and DNA attached to the gold surface at 5'-thiol termini. The presence of ssDNA and dsDNA in the monolayer facilitated the redox reaction of MB and DOX on the modified electrode. Both MB and DOX diffuse along the ssDNA in the ssDNA-containing monolayers, and they additionally intercalate into the dsDNA in the dsDNA-containing monolayers. No sufficient evidence is shown to indicate that an organized monolayer is formed by the thiol-labeled dsDNA on gold surface, and that the redox reactions of MB and DOX were carried out by electron transfer through DNA helix.     

Interaction of dimeric intercalating dyes with single-stranded DNA.

The unsymmetrical cyanine dye thiazole orange homodimer (TOTO) binds to single-stranded DNA (ssDNA, M13mp18 ssDNA) to form a fluorescent complex that is stable under the standard conditions of electrophoresis. The stability of this complex is indistinguishable from that of the corresponding complex of TOTO with double-stranded DNA (dsDNA). To examine if TOTO exhibits any binding preference for dsDNA or ssDNA, transfer of TOTO from pre-labeled complexes to excess unlabeled DNA was assayed by gel electrophoresis. Transfer of TOTO from M13 ssDNA to unlabeled dsDNA proceeds to the same extent as that from M13 dsDNA to unlabeled dsDNA. A substantial amount of the dye is retained by both the M13 ssDNA and M13 dsDNA even when the competing dsDNA is present at a 600-fold weight excess; for both dsDNA and ssDNA, the pre-labeled complex retains approximately one TOTO per 30 bp (dsDNA) or bases (ssDNA). Rapid transfer of dye from both dsDNA and ssDNA complexes is seen at Na+ concentrations > 50 mM. Interestingly, at higher Na+ or Mg2+ concentrations, the M13 ssDNA-TOTO complex appears to be more stable to intrinsic dissociation (dissociation in the absence of competing DNA) than the complex between TOTO and M13 dsDNA. Similar results were obtained with the structurally unrelated dye ethidium homodimer. The dsDNA- and ssDNA-TOTO complexes were further examined by absorption, fluorescence and circular dichroism spectroscopy. The surprising conclusion is that polycationic dyes, such as TOTO and EthD, capable of bis-intercalation, interact with dsDNA and ssDNA with very similar high affinity.     

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

The uvsX protein of bacteriophage T4 arranges single-stranded and double-stranded DNA into similar helical nucleoprotein filaments

The bacteriophage T4 uvsX gene codes for a DNA-binding protein that is important for genetic recombination in T4-infected cells. This protein is a DNA-dependent ATPase that resembles the Escherichia coli recA protein in many of its properties. We have examined the binding of purified uvsX protein to single-stranded DNA (ssDNA) and to double-stranded DNA (dsDNA) using electron microscopy to visualize the complexes that are formed and double label analysis to measure their protein content. We find that the uvsX protein binds cooperatively to dsDNA, forming filaments 14 nm in diameter with an apparently helical axial repeat of 12 nm. Each repeat contains about 42 base pairs and 9-12 uvsX protein monomers. In solutions containing Mg2+, the uvsX protein also binds cooperatively to ssDNA. The filaments that result are 14 nm in diameter, show a 12-nm axial repeat, and they are nearly identical in appearance to the filaments that contain dsDNA. In the filaments formed along ssDNA, each axial repeat contains about 49 DNA bases and 9-12 uvsX monomers. Both the filaments formed on the ssDNA and dsDNA show a strong tendency to align side-by-side. T4 gene 32 protein also binds cooperatively to ssDNA and interacts both physically and functionally with uvsX protein. However, when gene 32 and uvsX proteins were added to ssDNA together, no interaction between the two proteins was detected.     

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.     

The Effect of Mutation and Selection on Codon Adaptation in Escherichia coli Bacteriophage

Studying phage codon adaptation is important not only for understanding the process of translation elongation, but also for reengineering phages for medical and industrial purposes. To evaluate the effect of mutation and selection on phage codon usage, we developed an index to measure selection imposed by host translation machinery, based on the difference in codon usage between all host genes and highly expressed host genes. We developed linear and nonlinear models to estimate the C→T mutation bias in different phage lineages and to evaluate the relative effect of mutation and host selection on phage codon usage. C→T-biased mutations occur more frequently in single-stranded DNA (ssDNA) phages than in double-stranded DNA (dsDNA) phages and affect not only synonymous codon usage, but also nonsynonymous substitutions at second codon positions, especially in ssDNA phages. The host translation machinery affects codon adaptation in both dsDNA and ssDNA phages, with a stronger effect on dsDNA phages than on ssDNA phages. Strand asymmetry with the associated local variation in mutation bias can significantly interfere with codon adaptation in both dsDNA and ssDNA phages.     

Action of a mammalian AP-endonuclease on DNAs of defined sequences.

An apurinic/apyrimidinic (AP) specific endonuclease from mouse plasmacytoma cells (line MPC-11), was observed to cleave apurinic sites in oligonucleotides 9, 11, 12, 39 and 40 nucleotides in length. However, the enzyme failed to cleave AP-sites in two oligonucleotides 7 nucleotides in length. The maximum rates of digestion observed on these short single-stranded DNA (ssDNA) fragments were approximately 1/30 of the rates observed on double-stranded DNA (dsDNA). In studies using the Maxam-Gilbert DNA sequencing analysis, apurinic sites in purine-rich regions were preferentially cleaved in dsDNA but not in ssDNA, indicating that the enzyme has a sequence preference on dsDNA. These results suggest that some sites on DNA might be more efficiently repaired than others.     

Novel aspects of the structure of the Escherichia coli nucleoid investigated by a rapid sedimentation assay

The Escherichia coli nucleoid is maintained in its folded highly condensed state by constraints which involve RNA and protein. We have developed a rapid sedimentation assay to determine the state of folding of the membrane-free nucleoid. An approximate measure of the stability of the nucleoids under various conditions can then be estimated by measuring the temperature at which the nucleoids unfold. Using ethidium and gamma irradiation (which removes the negative supercoiling of the native nucleoid) as probes, it can be shown that there are two types of constraint involved in the condensation of the nucleoid. One of these constraints is destabilized by ethidium but stabilized by negative supercoiling; the second constraint is unaffected by both ethidium and negative supercoiling. Several models can be proposed: (i) a DNA . RNA duplex, (ii) a double-strand DNA (dsDNA) . RNA triplex, (iii) DNA-protein interactions, (iv) a topological knot with RNA, and (v) a DNA tetraplex. The topological knot model is not consistent with the data and many combinations of the others can be excluded. If RNA is involved in both constraints then RNA . DNA duplexes and dsDNA . RNA triplexes are involved in stabilizing the nucleoid.     

Homologous-pairing activity of the Bacillus subtilis bacteriophage SPP1 replication protein G35P

Genetic evidence suggests that the SPP1-encoded gene 35 product (G35P) is essential for phage DNA replication. Purified G35P binds single-strand DNA (ssDNA) and double-strand (dsDNA) and specifically interacts with SPP1-encoded replicative DNA helicase G40P and SSB protein G36P. G35P promotes joint molecule formation between a circular ssDNA and a homologous linear dsDNA with an ssDNA tail. Joint molecule formation requires a metal ion but is independent of a nucleotide cofactor. Joint molecules formed during these reactions contain a displaced linear ssDNA strand. Electron microscopic analysis shows that G35P forms a multimeric ring structure in ssDNA tails of dsDNA molecules and left-handed filaments on ssDNA. G35P promotes strand annealing at the AT-rich region of SPP1 oriL on a supercoiled template. These results altogether are consistent with the hypothesis that the homologous pairing catalyzed by G35P is an integral part of SPP1 DNA replication. The loading of G40P at a d-loop (ori DNA or at any stalled replication fork) by G35P could lead to replication fork reactivation.     

The N-terminal domain of Escherichia coli RecA have multiple functions in promoting homologous recombination

Escherichia coli RecA mediates homologous recombination, a process essential to maintaining genome integrity. In the presence of ATP, RecA proteins bind a single-stranded DNA (ssDNA) to form a RecA-ssDNA presynaptic nucleoprotein filament that captures donor double-stranded DNA (dsDNA), searches for homology, and then catalyzes the strand exchange between ssDNA and dsDNA to produce a new heteroduplex DNA. Based upon a recently reported crystal structure of the RecA-ssDNA nucleoprotein filament, we carried out structural and functional studies of the N-terminal domain (NTD) of the RecA protein. The RecA NTD was thought to be required for monomer-monomer interaction. Here we report that it has two other distinct roles in promoting homologous recombination. It first facilitates the formation of a RecA-ssDNA presynaptic nucleoprotein filament by converting ATP to an ADP-Pi intermediate. Then, once the RecA-ssDNA presynaptic nucleoprotein filament is stably assembled in the presence of ATPγS, the NTD is required to capture donor dsDNA. Our results also suggest that the second function of NTD may be similar to that of Arg243 and Lys245, which were implicated earlier as binding sites of donor dsDNA. A two-step model is proposed to explain how a RecA-ssDNA presynaptic nucleoprotein filament interacts with donor dsDNA.     

A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro

The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt. Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both [UvrD] and [DNA] under conditions such that UvrD-DNA binding is stoichiometric. Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths /=12 nt, and the unwinding amplitude displays a sigmoidal dependence on [UvrD(tot)]/[DNA(tot)]. Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro. Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP. Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.     

Modeling the early stage of DNA sequence recognition within RecA nucleoprotein filaments

Homologous recombination is a fundamental process enabling the repair of double-strand breaks with a high degree of fidelity. In prokaryotes, it is carried out by RecA nucleofilaments formed on single-stranded DNA (ssDNA). These filaments incorporate genomic sequences that are homologous to the ssDNA and exchange the homologous strands. Due to the highly dynamic character of this process and its rapid propagation along the filament, the sequence recognition and strand exchange mechanism remains unknown at the structural level. The recently published structure of the RecA/DNA filament active for recombination (Chen et al., Mechanism of homologous recombination from the RecA-ssDNA/dsDNA structure, Nature 2008, 453, 489) provides a starting point for new exploration of the system. Here, we investigate the possible geometries of association of the early encounter complex between RecA/ssDNA filament and double-stranded DNA (dsDNA). Due to the huge size of the system and its dense packing, we use a reduced representation for protein and DNA together with state-of-the-art molecular modeling methods, including systematic docking and virtual reality simulations. The results indicate that it is possible for the double-stranded DNA to access the RecA-bound ssDNA while initially retaining its Watson–Crick pairing. They emphasize the importance of RecA L2 loop mobility for both recognition and strand exchange.     

DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

Fluorescent Determination of Double-stranded DNA with an Anionic 1,1′-Azonaphthalene Derivative

Palatine chrome black 6BN (PCB6BN) is virtually non-fluorescent in an aqueous solution or in the presence of single-stranded DNA (ssDNA), whereas the fluorescence intensity of PCB6BN was linearly enhanced up to 300 μM of double-stranded DNA (dsDNA) base pairs. PCB6BN could be a useful fluorescent probe for quantifying dsDNA even when ssDNA is present for both heterogeneous and homogeneous assays.     

Electrophoretic Mobility of DNA in Solutions of High Ionic Strength

The free-solution mobilities of small single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) have been measured by capillary electrophoresis in solutions containing 0.01–1.0 M sodium acetate. The mobility of dsDNA is greater than that of ssDNA at all ionic strengths because of the greater charge density of dsDNA. The mobilities of both ssDNA and dsDNA decrease with increasing ionic strength until approaching plateau values at ionic strengths greater than ∼0.6 M. Hence, ssDNA and dsDNA appear to interact in a similar manner with the ions in the background electrolyte. For dsDNA, the mobilities predicted by the Manning electrophoresis equation are reasonably close to the observed mobilities, using no adjustable parameters, if the average distance between phosphate residues (the b parameter) is taken to be 1.7 Å. For ssDNA, the predicted mobilities are close to the observed mobilities at ionic strengths ≤0.01 M if the b-value is taken to be 4.1 Å. The predicted and observed mobilities diverge strongly at higher ionic strengths unless the b-value is reduced significantly. The results suggest that ssDNA strands exist as an ensemble of relatively compact conformations at high ionic strengths, with b-values corresponding to the relatively short phosphate-phosphate distances through space.     

Tyrosine phosphorylation enhances RAD52-mediated annealing by modulating its DNA binding

RAD52 protein has an important role in homology-directed DNA repair by mediating RAD51 nucleoprotein filament formation on single-stranded DNA (ssDNA) protected by replication protein-A (RPA) and annealing of RPA-coated ssDNA. In human, cellular response to DNA damage includes phosphorylation of RAD52 by c-ABL kinase at tyrosine 104. To address how this phosphorylation modulates RAD52 function, we used an amber suppressor technology to substitute tyrosine 104 with chemically stable phosphotyrosine analogue (p-Carboxymethyl-L-phenylalanine, pCMF). The RAD52(Y104pCMF) retained ssDNA-binding activity characteristic of unmodified RAD52 but showed lower affinity for double-stranded DNA (dsDNA) binding. Single-molecule analyses revealed that RAD52(Y104pCMF) specifically targets and wraps ssDNA. While RAD52(Y104pCMF) is confined to ssDNA region, unmodified RAD52 readily diffuses into dsDNA region. The Y104pCMF substitution also increased the ssDNA annealing rate and allowed overcoming the inhibitory effect of dsDNA. We propose that phosphorylation at Y104 enhances ssDNA annealing activity of RAD52 by attenuating dsDNA binding. Implications of phosphorylation-mediated activation of RAD52 annealing activity are discussed.     

Loading/release behavior of (chitosan/DNA)n layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage

In the present work, positively charged chitosan (CS) and negatively charged DNA were alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes, forming (CS/DNA)(n) layer-by-layer films. Cyclic voltammetry (CV) results showed that negatively charged electroactive probe, 9,10-anthraquinone-2,6-disulfonate (AQDS), could be loaded into the (CS/DNA)(n) films from its solution (1 mM at pH 7.0, containing 0.1 M NaCl), designated as (CS/DNA)(n)-AQDS, and then released from the films in blank buffers. The loading/release behavior of (CS/DNA)(n) films toward AQDS was found to be obviously different between double-stranded (dsDNA) and single-stranded DNA (ssDNA). The release rate of AQDS from (CS/dsDNA)(n) films was much slower than that from the ssDNA counterparts mainly because AQDS could be intercalated into the double helix structure of dsDNA despite the repulsion between likely charged AQDS and DNA. The loading/release behavior of (CS/DNA)(n) films toward AQDS in recognition of dsDNA and ssDNA was then successfully applied to electrochemically detect the damage of natural DNA caused by Fenton reaction. To further understand the essence of the interactions involved in the AQDS loading/release process for (CS/DNA)(n) films, comparison experiments were performed, in which either positively charged intercalator brilliant cresyl blue (BCB) was used to replace AQDS as the redox probe, or poly(diallyldimethylammonium) (PDDA) with relatively high positive charge density was used to replace CS as the constituent of layer-by-layer films with DNA. The loading/release behavior of DNA films toward electroactive intercalator may open new possibilities for dsDNA/ssDNA recognition and of DNA damage detection by electrochemistry.