The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair

The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied. It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA). The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha. The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA. The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein. The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase. A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.     

Participation of the SSB protein in the repair of the DNA of Ehrlich ascites carcinoma cells following UV irradiation

Synchronous changes were detected in the SSB-protein content of the chromatin and in the rate of repair DNA synthesis at different time intervals after UV-irradiation of Ehrlich ascites tumor cells. The amount of SSB-protein in the extra-chromatin fraction was in an inverse relation to its content in the chromatin, whereas the cumulative SSB-protein content remained invariable. Similar changes in the SSB-protein content of the chromatin and in repair synthesis were also registered after the effect of various doses of UV-light. The increase of the SSB-protein content in the chromatin was not connected with the postirradiation accumulation of single-strand sites in DNA.     

Reconstitution of the M13 major coat protein and its transmembrane peptide segment on a DNA template

The major coat protein (pVIII) of M13 phage is of particular interest to structure biologists since it functions in two different environments: during assembly and infection, it interacts with the bacterial lipid bilayer, but in the phage particle, it exists as a protein capsid to protect a closed circular, single-stranded DNA (ssDNA) genome. We synthesized pVIII and a 32mer peptide consisting of the transmembrane and DNA binding domains of pVIII. The 32mer peptide displays typically an alpha-helical structure in trifluroethanol or 0.2 M octylglucoside solutions similar to pVIII. Attachment of polyethylene glycol (PEG) onto the N-terminal of 32mer increased the alpha-helical content and the peptide thermal stability. The peptides were reconstituted with DNA from a detergent solution into a discrete (<200 nm diameter) nanoparticle on both linear double-stranded DNA (dsDNA) and linear ssDNA, where the linear dsDNA is used to mimic the closed circular, ssDNA in M13 phage, upon removal of the detergent. The peptide/DNA particle was an irregular and not a rod-shaped aggregate when imaged by atomic force microscopy. All three peptides underwent a structural transition from alpha-helix to beta-sheet within approximately 1 h of DNA addition to the detergent solution. There was a further decrease in alpha-helical content when the detergent was removed. The presence of anionic (such as octanoic acid) or cationic (such as 1,5-diaminopentane) molecules in the detergent mixture resulted in the retention of the peptide alpha-helical structure. Thus the interaction between the peptide and DNA in octylglucoside is driven by electrostatic forces, and peptide-peptide interactions are responsible for the transition from alpha-helix to beta-sheet conformation in pVIII and its analogues. These results suggest that the assembly process to form a rod-shaped phage is a delicate balance to maintain pVIII in an alpha-helical conformation that requires either an oriented bilayer to solubilize pVIII prior to interaction with the DNA or other phage proteins to nucleate pVIII in the alpha-helical conformation on the DNA.     

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.     

Participation of the SSB protein in the repair of the DNA in Ehrlich ascites carcinoma cells following gamma radiation

Survival of Ehrlich ascites tumor cells, SSB content of the chromatin, and repair DNA synthesis rate were investigated after gamma-irradiation, the rate of repair synthesis was shown to depend on the SSB-protein content of the chromatin. Changes in the amount of SSB-protein in the chromatin were not connected with the postirradiation accumulation of single-strand sites in DNA.     

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.     

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.     

Use of benzoyl-naphthyl-DEAE-cellulose chromatography for determining the ability of proteins from Ehrlich ascites carcinoma, binding with single-stranded DNA (SSB-proteins), to destabilize the DNA double helix]

Single-stranded DNA-binding proteins (SSB-proteins) isolated from Ehrlich ascites tumour (EAT) cells were incubated for 30 min at 5 mM NaCl with salmon sperm DNA or [3H]DNA from EAT at the SSB-protein/DNA ratio (w/w) of 0 to 4.5. After addition of sodium dodecyl sulfate up to a 0.05% concentration, the proteins were applied to columns with benzoylated naphthoylated DEAE-cellulose. Double-stranded DNA was eluted by 1 M NaCl; the DNA containing single-stranded regions was eluted by 50% dimethylformamide. There was a progressive lowering of the DNA content in the first eluate and a rise in the second eluate, as could be evidenced from the increase in the SSB-protein/DNA w/w ratio. This effect was more pronounced in the case of homologous DNA and was not coupled with the nuclease activity of SSB proteins. It was concluded that EAT SSB-proteins are "DNA-unwinding" proteins.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.     

Kinetic estimation of the base sequence complexity of poly(A)-containing mRNA in Ehrlich-ascites-tumor cells and its abundance in nuclear RNA.

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27,000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)-containing mRNA up to an rot of 3,000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity.     

Interaction of the HMG1 protein with nucleic acids

Binding constants have been measured for the interaction of the protein HMG1 with native DNA, denatured DNA and a number of polynucleotides at near-physiological ionic strengths, using gel filtration and thermal denaturation. The interaction of HMG1 with DNA is shown to be noncooperative and reversible. Nucleic acids form the following series in order of increasing binding constants: poly(U) integral of poly(A) less than poly(dA) less than dsDNA integral of poly(dA) X poly(dT) integral of poly(dG) X poly(dC) much less than poly[d(A-T]) integral of ssDNA.     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

Three-stranded paranemic joints: architecture, topological constraints and movement

The RecA and SSB proteins will catalyze the joining of two DNA molecules containing homologous sequences but lacking homologous ends in a reaction termed paranemic joining. The absence of homologous ends can be achieved by (1) pairing two circular DNAs or (2) using linear DNA(s) with ends lacking homology to the pairing partner. Here we have used electron microscopy (EM) to examine such pairings. Circular M13 single-stranded (ss) DNA enveloped by RecA protein into a presynaptic filament was paired with linear M13mp7 double-stranded (ds) DNA containing non-M13 sequences at its ends. Joint complexes were frequently seen in which the dsDNA was joined with the presynaptic filament over several kilobase (10(3) bases) lengths of the dsDNA. In this region, the presynaptic filament appeared disorganized as contrasted to the customary helical structure of the filament containing only a single strand of DNA. The same ultrastructure, but with greater detail, was observed when the samples were prepared for EM without fixation using a new method of fast-freezing and freeze-drying. EM immunogold staining demonstrated the presence of SSB protein in the disorganized region containing all three strands, but not in the regular helically arranged region. Psoralen photo-crosslinking of the DNA in the joint complexes revealed that the three DNA strands were in close proximity only over a single short (200 to 300 base-pairs) region. The joining of nicked circular M13 dsDNA and presynaptic filaments containing circular M13 ssDNA resulted in the intertwining of the dsDNA about the circular presynaptic filament. The joints produced in this case were short, as was the single region of psoralen photo-crosslinking of the three DNA strands. A model of how these long three-stranded joints form is presented involving the movement of a short "true" paranemic joint along the presynaptic filament.     

The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells

Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied. There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9). It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis. It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.     

Strand displacement of double-stranded DNA by triplex-forming antiparallel purine-hairpins

We characterize the binding affinity and the thermodynamics of hybridization of triplex-forming antiparallel purine-hairpins composed of two antiparallel purine domains linked by a loop directed toward single-stranded and double-stranded DNA (ssDNA, dsDNA). Gel retardation assays and melting experiments reveal that a 13-mer purine-hairpin binds specifically and with a K ( d ) of 8 x 10(8) M to polypyrimidine ssDNA to form a triple helical structure. Remarkably, we show that purine-hairpins also bind polypurine/polypyrimidine stretches included in a dsDNA of several hundred bp in length. Binding of purine-hairpins to dsDNA occurs by triplex formation with the polypyrimidine strand, causing displacement of the polypurine strand. Because triplex formation is restricted to polypurine/polypyrimidine stretches of dsDNA, we studied the triplex formation between purine-hairpins and polypyrimidine targets containing purine interruptions. We found that an 11-mer purine-hairpin with an adenine opposite to a guanine interruption in the polypyrimidine track binds to ssDNA and dsDNA, allowing expansion of the possible target sites and increase in the length of purine-hairpins. Thus, when using a 20-mer purine-hairpin targeting an interruption-containing polypyrimidine target, the binding affinity is increased compared to its 13-mer antiparallel purine-hairpin counterpart. Surprisingly, this increase is much more pronounced than that observed for a tail-clamp purine-hairpin extended up to 20 nt in the Watson-Crick domain only. Thus, triplexforming antiparallel purine-hairpins can be a potentially useful strategy for both single-strand and double-strand nucleic acid recognition.     

Binding and reversible denaturation of double-stranded DNA by Ff gene 5 protein

The gene 5 protein (g5p) from Ff filamentous virus is a model single-stranded DNA (ssDNA) binding protein that has an oligonucleotide/oligosaccharide binding (OB)-fold structure and binding properties in common with other ssDNA-binding proteins. In the present work, we use circular dichroism (CD) spectroscopy to analyze the effects of amino acid substitutions on the binding of g5p to double-stranded DNA (dsDNA) compared to its binding to ssDNA. CD titrations of poly[d(A). d(T)] with mutants of each of the five tyrosines of the g5p showed that the 229-nm CD band of Tyr34, a tyrosine at the interface of adjacent protein dimers, is reversed in sign upon binding to the dsDNA, poly[d(A). d(T)]. This effect is like that previously found for g5p binding to ssDNAs, suggesting there are similarities in the protein-protein interactions when g5p binds to dsDNA and ssDNA. However, there are differences, and the possible perturbation of a second tyrosine, Tyr41, in the complex with dsDNA. Three mutant proteins (Y26F, Y34F, and Y41H) reduced the melting temperature of poly[d(A). d(T)] by 67 degrees C, but the wild-type g5p only reduced it by 2 degrees C. This enhanced ability of the mutants to denature dsDNA suggests that their binding affinities to dsDNA are reduced more than are their binding affinities to ssDNA. Finally, we present evidence that when poly[d(A). d(T)] is melted in the presence of the wild-type, Y26F, or Y34F proteins, the poly[d(A)] and poly[d(T)] strands are separately sequestered such that renaturation of the duplex is facilitated in 2 mM Na(+).     

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.     

SOS repair can be about as effective for single-stranded DNA as for double-stranded DNA and even more so.

As ordinarily measured, the SOS repair of damaged DNA by Weigle reactivation appears to be more effective for double-stranded (ds) than for single-stranded (ss) DNA bacteriophages. A complicating feature, which is usually not considered, is the possibility of DNA-protein cross-linking of ssDNA to the viral capsid, which would conceivably be an extraneous source of nonreactivable lesions. This idea is supported in studies of phage S13 by the observation that photoreactivation more than doubles when naked ssDNA is substituted for encapsidated ssDNA as the UV target. The same effect was observed for Weigle reactivation; there was little, if any, difference in the reactivation of ssDNA and dsDNA when naked DNA was irradiated. Moreover, in a uvrA mutant, ssDNA actually had the advantage; Weigle reactivation was then more than twice as effective for ssDNA as for dsDNA. It is also shown that when a suitable measure of Weigle mutagenesis is used, there is no convincing evidence that dsDNA is mutagenized more effectively than ssDNA.     

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.