dsDNA Oligomers Research Articles

Electrophoretically Snagging Viral Genomes in Wormlike Micelle Networks Using Peptide Nucleic Acid Amphiphiles and dsDNA Oligomers.

We demonstrate that the attachment of 30-170 bp dsDNA oligomers to ssDNA viral genomes gives a significant additional mobility shift in micelle-tagging electrophoresis (MTE). In MTE, a modified peptide nucleic acid amphiphile is attached to the viral genome to bind drag-inducing micelles present in capillary electrophoresis running buffers. Further attachment of 30-170 bp dsDNA oligomers drastically shifts the mobility of the 5.1 kB ssDNA genome of mouse minute virus (MMV), providing a new mechanism to improve resolution in CE-based analysis of kilobase nucleic acids. A model based on biased-reptation electrophoresis, end-labeled free-solution electrophoresis, and Ferguson gel-filtration theory is presented to describe the observed mobility shifts.

Abstract 3113: Development of HTS enzymatic assays for WRN helicase and exonuclease functions

Abstract WRN, a double strand break repair enzyme with ATP-dependent DNA-helicase and 3’- >5’ exonuclease activities, is under intense focus as a therapeutic target because of its synthetic lethality in tumors with microsatellite instability. To accelerate discovery of WRN inhibitors, we developed and validated homogenous, fluorescence-based high throughput assays for WRN helicase and exonuclease enzymatic activities using the Transcreener HTS Assay platform, which relies on highly selective antibodies to detect nucleotides in a homogenous (mix-and-read) format with far red fluorescent readouts. We produced the WRN helicase domain (aa 500-946, N-terminal 6xHis) in BaV-infected insect cells and optimized detection of its dsDNA-dependent ATPase activity using the Transcreener ADP2 Assay in FP, TR-FRET, and FI detection modes. Optimal reaction conditions included 50 μM ATP (approximate Km) and saturating concentration (100 nM) of a 3’ flapped duplex DNA oligomer; these conditions yielded a good signal with 250 pM WRN, a kcat of 300 min−1, and a Z’ of 0.85. An IC50 of 35 nM was determined for the probe inhibitor NSC-617145, and a 1,280 compound pilot screen (Tocris 2.0 Micro) identified multiple hits that were confirmed in dose response experiments. We produced the WRN 3’-5’ exonuclease domain (aa 1-133-6xHis) in E. coli and used the Transcreener dAMP Exonuclease Assay to allow direct, FP-based detection of dAMP monomers released from a 52 bp dsDNA oligomer with a 5’ overhang. With saturating DNA present (1 μM), the assay produced a good signal with 1 nM enzyme; the Z’ value was 0.89. There are no published inhibitors for the WRN exonuclease, but suramin inhibited with an IC50 of approximately 100 nM. These assays allow sensitive, robust, quantitative detection of WRN ATPase and exonuclease activities using an extensively validated HTS platform; they should be valuable tools for screening and optimizing small molecules targeting specific WRN functions. Citation Format: Ha Pham, Mahbbat Ali, Anibal Ramos Martinez, Robert Lowery. Development of HTS enzymatic assays for WRN helicase and exonuclease functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3113.

Ammonium Gemini Surfactants Form Complexes with Model Oligomers of siRNA and dsDNA.

Dimeric cationic surfactants (gemini-type) are a group of amphiphilic compounds with potential use in gene therapy as effective carriers for nucleic acid transfection (i.e., siRNA, DNA, and plasmid DNA). Our studies have shown the formation of lipoplexes composed of alkanediyl-α,ω-bis[(oxymethyl)dimethyldodecylammonium] chlorides and selected 21-base-pair nucleic acid (dsDNA and siRNA) oligomers. To examine the structure and physicochemical properties of these systems, optical microscopy, circular dichroism spectroscopy (CD), small-angle X-ray scattering of synchrotron radiation (SR-SAXS), and agarose gel electrophoresis (AGE) were used. The lengths of spacer groups of the studied surfactants had a significant influence on the surfactants’ complexing properties. The lowest charge ratio (p/n) at which stable lipoplexes were observed was 1.5 and the most frequently occurring microstructure of these lipoplexes were cubic and micellar phases for dsDNA and siRNA, respectively. The cytotoxicity tests on HeLa cells indicated the non-toxic concentration of surfactants to be at approximately 10 µM. The dicationic gemini surfactants studied form complexes with siRNA and dsDNA oligomers; however, the complexation process is more effective towards siRNA. Therefore these systems could be applied as transfection systems for therapeutic nucleic acids.

Monitoring Base-Specific Dynamics during Melting of DNA-Ligand Complexes Using Temperature-Jump Time-Resolved Infrared Spectroscopy.

Ultrafast time-resolved infrared spectroscopy employing nanosecond temperature-jump initiation has been used to study the melting of double-stranded (ds)DNA oligomers in the presence and absence of minor groove-binding ligand Hoechst 33258. Ligand binding to ds(5'-GCAAATTTCC-3'), which binds Hoechst 33258 in the central A-tract region with nanomolar affinity, causes a dramatic increase in the timescales for strand melting from 30 to ∼250 μs. Ligand binding also suppresses premelting disruption of the dsDNA structure, which takes place on 100 ns timescales and includes end-fraying. In contrast, ligand binding to the ds(5'-GCATATATCC-3') sequence, which exhibits an order of magnitude lower affinity for Hoechst 33258 than the A-tract motif, leads to an increase by only a factor of 5 in melting timescales and reduced suppression of premelting sequence perturbation and end-fraying. These results demonstrate a dynamic impact of the minor groove ligand on the dsDNA structure that correlates with binding strength and thermodynamic stabilization of the duplex. Moreover, the ability of the ligand to influence base pairs distant from the binding site has potential implications for allosteric communication mechanisms in dsDNA.

Chemical cross‐linking of doxorubicin to synthetic oligonucleotides by formaldehyde

Anthacycline antibiotics, such as doxorubicin and daunorubicin, are among the most effective anti‐cancer drugs that treat various cancers. Previous research has shown that doxorubicin and daunorubicin were efficiently cross‐linked to double‐stranded (ds) DNA in the presence of formaldehyde in a regioselective and base specific manner. In an effort to further explore the cross‐linking of anthracycline antibiotics to DNA, doxorubicin is crosslinked to a variety of synthetic oligonucleotides including sequence specific ds and single stranded (ss) DNA oligomers. Our results demonstrate that doxorubicin is able to efficiently crosslink to the N2 amino group of guanine for the ds DNA oligomers. Intriguingly, we also discovered that doxorubicin is able to crosslink to ssDNA and ssRNA specifically to guanine residues by formaldehyde. These results suggest that ssDNA and ssRNA may serve as a new delivery system for the treatment of cancer patients by doxorubicin and daunorubicin.Support or Funding Information1R15GM109254‐01A1; R25GM061347

Thermodynamics for the Formation of Double-Stranded DNA-Single-Walled Carbon Nanotube Hybrids.

For the first time, the thermodynamics are described for the formation of double-stranded DNA (ds-DNA)-single-walled carbon nanotube (SWNT) hybrids. This treatment is applied to the exchange reaction of sodium cholate (SC) molecules on SWNTs and the ds-DNAs d(A)20 -d(T)20 and nuclear factor (NF)-κB decoy. UV/Vis/near-IR spectroscopy with temperature variations was used for analyzing the exchange reaction on the SWNTs with four different chiralities: (n,m)=(8,3), (6,5), (7,5), and (8,6). Single-stranded DNAs (ss-DNAs), including d(A)20 and d(T)20, are also used for comparison. The d(A)20-d(T)20 shows a drastic change in its thermodynamic parameters around the melting temperature (Tm ) of the DNA oligomer. No such Tm dependency was measured, owing to high Tm in the NF-κB decoy DNA and no Tm in the ss-DNA.

Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

The Cooperative Assembly of IFI16 Filaments on dsDNA Provides Insights into Host Defense Strategy

Whether host DNA-receptors have any capacity to distinguish self from nonself at the molecular level is one of the foremost questions in the innate immunity of mammals. By using quantitative assays and electron microscopy, we show that cooperatively assembling into filaments on dsDNA may serve an integral mechanism by which human interferon inducible protein 16 (IFI16) engages foreign DNA. IFI16 is essential for defense against a number of different pathogens, and its aberrant activity is also implicated in several autoimmune disorders such as Sjögren's syndrome. IFI16 cooperatively binds dsDNA in a length-dependent manner and clusters into distinct protein filaments even in the presence of excess dsDNA. Consequently, the assembled IFI16•dsDNA oligomers are clearly different from the previously proposed noninteracting entities resembling beads on a string. The isolated DNA-binding domains of IFI16 engage dsDNA without forming filaments and with weak affinity, and it is the non-DNA binding pyrin domain (PYD) of IFI16 that drives the cooperative filament assembly. The surface residues on the PYD that mediate the cooperative DNA-binding are conserved, suggesting that related receptors use a common mechanism. These results suggest that IFI16 clusters into signaling complexes in a switch-like manner, and that it may use the size of naked dsDNA as molecular ruler to distinguish self from nonself.

The free solution mobility of DNA and other analytes varies as the logarithm of the fractional negative charge

The free solution mobilities of ssDNA and dsDNA molecules with variable charge densities have been measured by CE. DNA charge density was modified either by appending positively or negatively charged groups to the thymine residues in a 98 bp DNA molecule, or by replacing some of the negatively charged phosphate internucleoside linkers in small ssDNA or dsDNA oligomers with positively charged phosphoramidate linkers. Mobility ratios were calculated for each dataset by dividing the mobility of a charge variant by the mobility of its unmodified parent DNA. Mobility ratios essentially eliminate the effect of the BGE on the observed mobility, making it possible to compare analytes measured under different experimental conditions. Neutral moieties attached to the thymine residues in the 98-bp DNA molecule had little or no effect on the mobility ratios, indicating that bulky substituents in the DNA major groove do not affect the mobility significantly. The mobility ratios observed for the thymine-modified and linker-modified DNA charge variants increased approximately linearly with the logarithm of the fractional negative charge of the DNA. Mobility ratios calculated from previous studies of linker-modified DNA charge variants and small multicharged organic molecules also increased approximately linearly with the logarithm of the fractional negative charge of the analyte. The results do not agree with the Debye-Hückel-Onsager theory of electrophoresis, which predicts that the mobility of an analyte should depend linearly on analyte charge, not the logarithm of the charge, when the frictional coefficient is held constant.

Reaction mechanism of Ru(II) piano-stool complexes: umbrella sampling QM/MM MD study.

Biologically relevant interactions of piano-stool ruthenium(II) complexes with ds-DNA are studied in this article by hybrid quantum mechanics-molecular mechanics (QM/MM) computational technique. The whole reaction mechanism is divided into three phases: (i) hydration of the [Ru(II) (η(6) -benzene)(en)Cl](+) complex, (ii) monoadduct formation between the resulting aqua-Ru(II) complex and N7 position of one of the guanines in the ds-DNA oligomer, and (iii) formation of the intrastrand Ru(II) bridge (cross-link) between two adjacent guanines. Free energy profiles of all the reactions are explored by QM/MM MD umbrella sampling approach where the Ru(II) complex and two guanines represent a quantum core, which is described by density functional theory methods. The combined QM/MM scheme is realized by our own software, which was developed to couple several quantum chemical programs (in this study Gaussian 09) and Amber 11 package. Calculated free energy barriers of the both ruthenium hydration and Ru(II)-N7(G) DNA binding process are in good agreement with experimentally measured rate constants. Then, this method was used to study the possibility of cross-link formation. One feasible pathway leading to Ru(II) guanine-guanine cross-link with synchronous releasing of the benzene ligand is predicted. The cross-linking is an exergonic process with the energy barrier lower than for the monoadduct reaction of Ru(II) complex with ds-DNA.

Formation of S–Cl Phosphorothioate Adduct Radicals in dsDNA S-Oligomers: Hole Transfer to Guanine vs Disulfide Anion Radical Formation

In phosphorothioate-containing dsDNA oligomers (S-oligomers), one of the two nonbridging oxygen atoms in the phosphate moiety of the sugar-phosphate backbone is replaced by sulfur. In this work, electron spin resonance (ESR) studies of one-electron oxidation of several S-oligomers by Cl2(•-) at low temperatures are performed. Electrophilic addition of Cl2(•-) to phosphorothioate with elimination of Cl(-) leads to the formation of a two-center three-electron σ(2)σ*(1)-bonded adduct radical (-P-S-̇Cl). In AT S-oligomers with multiple phosphorothioates, i.e., d[ATATAsTsAsT]2, -P-S-̇Cl reacts with a neighboring phosphorothioate to form the σ(2)σ*(1)-bonded disulfide anion radical ([-P-S-̇S-P-](-)). With AT S-oligomers with a single phosphorothioate, i.e., d[ATTTAsAAT]2, reduced levels of conversion of -P-S-̇Cl to [-P-S-̇S-P-](-) are found. For guanine-containing S-oligomers containing one phosphorothioate, -P-S-̇Cl results in one-electron oxidation of guanine base but not of A, C, or T, thereby leading to selective hole transfer to G. The redox potential of -P-S-̇Cl is thus higher than that of G but is lower than those of A, C, and T. Spectral assignments to -P-S-̇Cl and [-P-S-̇S-P-](-) are based on reaction of Cl2(•-) with the model compound diisopropyl phosphorothioate. The results found for d[TGCGsCsGCGCA]2 suggest that [-P-S-̇S-P-](-) undergoes electron transfer to the one-electron-oxidized G, healing the base but producing a cyclic disulfide-bonded backbone with a substantial bond strength (50 kcal/mol). Formation of -P-S-̇Cl and its conversion to [-P-S-̇S-P-](-) are found to be unaffected by O2, and this is supported by the theoretically calculated electron affinities and reduction potentials of [-P-S-S-P-] and O2.

Helical Structure Determines Different Susceptibilities of dsDNA, dsRNA, and tsDNA to Counterion-Induced Condensation

Recent studies of counterion-induced condensation of nucleic acid helices into aggregates produced several puzzling observations. For instance, trivalent cobalt hexamine ions condensed double-stranded (ds) DNA oligomers but not their more highly charged dsRNA counterparts. Divalent alkaline earth metal ions condensed triple-stranded (ts) DNA oligomers but not dsDNA. Here we show that these counterintuitive experimental results can be rationalized within the electrostatic zipper model of interactions between molecules with helical charge motifs. We report statistical mechanical calculations that reveal dramatic and nontrivial interplay between the effects of helical structure and thermal fluctuations on electrostatic interaction between oligomeric nucleic acids. Combining predictions for oligomeric and much longer helices, we also interpret recent experimental studies of the role of counterion charge, structure, and chemistry. We argue that an electrostatic zipper attraction might be a major or even dominant force in nucleic acid condensation.

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon.

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to single-stranded DNA (ssDNA) with a site size of ∼25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.

Energetics of the E. Coli Pria Helicase Interactions with the Double Stranded DNA

Quantitative analyses of the interaction of the Escherichia coli monomeric PriA helicase with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence resonance energy transfer methods. The experiments have been performed with different dsDNA oligomers, long enough, to encompass the total DNA-binding site, as well as the DNA-binding site proper of the enzyme. Interactions with the dsDNA oligomers were examined as a function of different temperature, salts, and nucleotide cofactors. The stoichimetry of the PriA helicase - dsDNA is different from the stoichiometry of the analogous complexes with the ss conformation of the nucleic acid, indicating a very different orientation of the helicase on the dsDNA. Surprisingly, the intrinsic dsDNA-affinity of the enzyme is dramatically higher than the ssDNA affinity, indicating strong selectivity of the helicase for the dsDNA conformation of the nucleic acid. The intrinsic affinities are salt-dependent and the formation of the PriA helicase - dsDNA complex is accompanied by a net ion change. Moreover, the presence of nucleotide cofactors has a profound effect on the dsDNA interactions of the enzyme with the DNA. The interactions of the PriA helicase with the dsDNA are characterized by very weak, if any, cooperative interactions. The significance of these results on activities of the PriA helicase in the cell metabolism is discussed.

Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery

Ionically crosslinked nanoparticles based on high and low molecular weight chitosans (CS) were formulated with plasmid DNA or dsDNA oligomers using the ionic gelation technique with pentasodium tripolyphospate (TPP) as crosslinking agent. The resulting CS/TPP nanoparticles were investigated with regard to their physical–chemical properties, in vitro transfection efficiency, toxicity, cellular uptake, and in vivo gene expression following intratracheal administration to mice. The effects of co-formulating the nanoparticles with a model protein, BSA, were also studied. CS/TPP nanoparticles showed high encapsulation efficiencies both for plasmid DNA and dsDNA oligomers (20-mers), independent of CS molecular weight. TEM images revealed a spherical shape of the CS/TPP nanoparticles in contrast to the heterogeneous and irregular morphology displayed by conventional chitosan polyplexes. The nanoparticles showed high physical stability and no DNA release could be detected in diverse release media, nor even after incubation with heparin. Low molecular weight (LMW) CS/TPP nanoparticles gave high gene expression levels in HEK 293 cells already 2 days after transfection, reaching a plateau of sustained and high gene expression between 4 and 10 days. The inclusion of BSA into the nanostructures did not alter the inherent transfection efficiency of the nanoparticles. Confocal studies suggest endocytotic cellular uptake of the nanoparticles and a subsequent release into the cytoplasm within 14 h. LMW CS/TPP nanoparticles mediated a strong beta-galactosidase expression in vivo after intratracheal administration. The results of this study forward ionically crosslinked CS/TPP nanoparticles as a biocompatible non-viral gene delivery system and generate a solid ground for further optimization studies, for example with regard to steric stabilization and targeting.

Direct Observation of the Hole Protonation State and Hole Localization Site in DNA-Oligomers

In this work, it is shown that the incorporation of an 8-deuteroguanine (G*) moiety in DNA-oligomers allows for direct determination at 77 K of (i) the location of holes (i.e., the radical site) within dsDNA at specific base sites, even within stacks of G, as well as (ii) the protonation state of the hole at that site. These findings are based on our work and demonstrate that selective deuteration at C-8 on a guanine moiety in dGuo results in an ESR signal from the guanine cation radical (G**(+)) which is easily distinguishable from that of the undeuterated guanine cation radical (G*(+)). G**(+) is also found to be easily distinguishable from its conjugate base, the N1-deprotonated radical, G*(-H)*. Our ESR results clearly establish that at 77 K (i) one-electron oxidized guanine in double stranded DNA-oligomers exists as the deprotonated neutral radical G(-H)* as a result of facile proton transfer to the hydrogen bonded cytosine and (ii) the hole is preferentially located at the 5'-end in several ds DNA-oligomers with a GGG sequence.

Phosphorothioation of Oligonucleotides Strongly Influences the Inhibition of Bacterial (M.HhaI) and Human (Dnmt1) DNA Methyltransferases

Methyltransferase inhibitors: Short double-stranded oligonucleotides that have a hemimethylated target sequence and 5-fluoro-2'-deoxycytidine as a suicide inhibitor as well as their phosphorothioated analogues were tested for their ability to inhibit the bacterial methyltransferase M.HhaI and the human Dnmt1 in vitro.The cytidine analogue 5-fluoro-2'-deoxycytidine (dC(F)) is a mechanism-based inhibitor of DNA methyltransferases. We report the synthesis of short 18-mer dsDNA oligomers containing a triple-hemimethylated CpG motive as a recognition sequence for the human methyltransferase Dnmt1. The DNA strands carry within these CpG islands dC(F) building blocks that function as mechanism-based inhibitors of the analyzed methyltransferases. In addition, we replaced the phosphodiester backbones at defined positions by phosphorothioates. These hypermodified DNA strands were investigated as inhibitors of the DNA methyltransferases M.HhaI and Dnmt1 in vitro. We could show that both methylases behave substantially differently in respect to the amount of DNA backbone modification.

Synthesis and properties of oligodeoxynucleotides incorporating 5-carboxydeoxycytidine

We synthesized oligodeoxynucleotide derivatives containing 5-carboxydeoxycytidine (1) as a new deoxynucleoside derivative having an anionic residue at the base part. The base pairing properties of the 5-carboxycytosine base were evaluated by use of ab initio calculations at the MP2/6-31G++(2d,p)//HF/6-31G(d) level. These calculations suggest that the Hoogsteen-type hydrogen bond in DNA triplex is less stable than that of the canonical C(+)-G pair and that the Watson-Crick base pair of this modified base with G is slightly more stable than the natural G-C base pair. These expectations were confirmed by T(m) experiments of duplexes and triplexes formed by hybridization of an oligodeoxynucleotide 14mer incorporating 5-carboxydeoxycytidine with ssDNA and dsDNA oligomers, respectively.

Interactions of the DNA Polymerase X of African Swine Fever Virus with Double-stranded DNA. Functional Structure of the Complex

Interactions of the polymerase X of African swine fever virus with the double-stranded DNA (dsDNA) have been studied with fluorescent dsDNA oligomers, using quantitative fluorescence titrations, analytical ultracentrifugation, and fluorescence energy transfer techniques. Studies with unmodified dsDNAs were performed, using competition titration method. ASV pol X binds the dsDNA with a site-size of n = 10(±2) base-pairs, which is significantly shorter than the total site-size of 16(±2) nucleotides of the enzyme–ssDNA complex. The small site size indicates that the enzyme binds the dsDNA exclusively using the proper DNA-binding subsite. Fluorescence energy transfer studies between the tryptophan residue W92 and the acceptor, located at the 5′ or 3′ end of the dsDNA, suggest strongly that the proper DNA-binding subsite is located on the non-catalytic C-terminal domain. Moreover, intrinsic interactions with the dsDNA 10-mer or 20-mer are accompanied by the same net number of ions released, independent of the length of the DNA, indicating the same length of the DNA engaged in the complex. The dsDNA intrinsic affinity is about two orders of magnitude higher than the ssDNA affinity, indicating that the proper DNA-binding subsite is, in fact, the specific dsDNA-binding site. Surprisingly, ASFV pol X binds the dsDNA with significant positive cooperativity, which results from protein–protein interactions. Cooperative interactions are accompanied by the net ion release, with anions participating in the ion-exchange process. The significance of these results for ASFV pol X activity in the recognition of damaged DNA is discussed.