Spontaneous Cleavage of Single and Double Stranded 4′-DNA Radicals Under Aerobic Conditions

Abstract

The O₂-induced strand scission of 4′-DNA radicals is initiated by a reversible O₂ addition reaction. The rate coefficient of the O₂ release from the 4′-DNA peroxyl radical is 1.00 s^?1 in single strands and 0.05 s^?1 in double strands at 20°C. Because of this reversibility, an O₂-dependent strand cleavage occurs only in the presence of H-donors which trap the 4′-DNA peroxyl radicals yielding DNA hydroperoxides. At very low H-donor concentrations the strand scission is the result of an O₂-independent, spontaneous reaction even under aerobic conditions.     

Study of helix-coil transition of DNA by dielectric constant measurement

The thermal helix–coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix–coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix–coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix–coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.     

Relaxation kinetics of the triple-stranded helix-coil transition at short chain length. A model including the staggering of chains.

The relaxation kinetics of a staggering zipper model are presented, in which the formation of helical nuclei is regarded to be rate-limiting and in which the sliding of strands along the helices is prohibited. Instead a realignment of staggered chains is only possible via complete uncoiling. While maintaining the third order of the initial reaction as in an all-or-none mechanism, the model predicts a large range of relaxation times, which contribute to the mean relaxation time according to the stability of the individual species and which are weakly coupled in the range of small amounts of helical content. The model can easily be compared to experimental results and agrees well with relaxation data obtained from a triple-helical peptide fragment of collagen. It may be readily expanded to other multistranded helix ? coil transitions with steady-state formation of the individual species and it suggests that the fraying of the helix ends is hidden by the fast-relaxation times due to the equilibration of the shortest helical species.     

The strand-separation transition of T2 bacteriophage DNA

Strand separation of T2 DNA has been investigated in a helix-destabilizing solvent. Temperature-shift experiments in which the conformation of the DNA is monitored by its viscosity, sedimentation behavior, and kinetics of helix formation show that a well-defined strand-separation transition follows the helix–coil transition usually observed by changes in absorbance. For T2 DNA, this strand-separation transition is 70% as broad as the helix–coil transition, and is characterized by extremely slow kinetics of conformational change in the population. Strand separation requires the expansion of the two-stranded coil observed at the end of the helix–coil transition. This expansion is apparently coupled with the disurption of the last remaining base pairs in the molecule. The expansion process increases the viscosity, and can be readily followed as a function of time and/or temperature. Subsequent separation of the expanded form into complementary strands results in a viscosity decrease, the net result of a reduction in hydrodynamic volume and the halving of the molecular weight. Only under conditions where the driving force for strand separation is large are these events at all synchronous in the population. When the kinetics of conformational change are complete in the strand-separation transition, a mixture of expanded forms and separate strands is observed; the breadth of the transition reflects differences in stability with respect to strand separation among the molecules in the population. The transition exhibits hysteresis and is not a reversible equilibrium between double-stranded and single-stranded forms. It appears that renucleation is kinetically forbidden within the strand-separation region.     

Artificial restriction DNA cutter for site-selective scission of double-stranded DNA with tunable scission site and specificity

The artificial restriction DNA cutter (ARCUT) method to cut double-stranded DNA at designated sites has been developed. The strategy at the base of this approach, which does not rely on restriction enzymes, is comprised of two stages: (i) two strands of pseudo-complementary peptide nucleic acid (pcPNA) anneal with DNA to form 'hot spots' for scission, and (ii) the Ce(IV)/EDTA complex acts as catalytic molecular scissors. The scission fragments, obtained by hydrolyzing target phosphodiester linkages, can be connected with foreign DNA using DNA ligase. The location of the scission site and the site-specificity are almost freely tunable, and there is no limitation to the size of DNA substrate. This protocol, which does not include the synthesis of pcPNA strands, takes approximately 10 d to complete. The synthesis and purification of the pcPNA, which are covered by a related protocol by the same authors, takes an additional 7 d, but pcPNA can also be ordered from custom synthesis companies if necessary.     

Fabrication of DNA/RNA Hybrids Through Sequence-Specific Scission of Both Strands by pcPNA-S1 Nuclease Combination

By combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with S1 nuclease, a tool for site-selective and dual-strand scission of DNA/RNA hybrids has been developed. Both of the DNA and the RNA strands in the hybrids are hydrolyzed at desired sites to provide unique sticky ends. The scission fragments are directly ligated with other DNA/RNA hybrids by using T4 DNA ligase, resulting in the formation of desired recombinant DNA/RNA hybrids.     

Elongational flow studies on DNA in aqueous solution and stress-induced scission of the double helix.

Elongational flow techniques are used to investigate the birefringent response and flow-induced molecular scission of monodisperse phage-DNA samples in aqueous solution. A 4-roll mill apparatus was used to characterize the solutions at low stain rates, epsilon less than or equal to 300 s-1, and the opposed jets apparatus used to study fracture of the DNA molecules at strain rates up to 15 x 10(3) s-1. The molecular weight values were measured before and after fracture in elongational flow using the high-resolution technique of pulsed field gel electrophoresis (PFGE). The birefringent response incorporates both rigid and flexible components. The birefringence is nonlocalized and rises gradually to a plateau value, similar to rigid-rod behavior. In addition a certain minimum value in the strain rate is necessary, an onset value epsilon 0, before the signal appears, indicating a flexible component. This behavior is consistent with a hinged-rod model and is similar to that observed for the protein collagen molecule at elevated temperature. We propose that this type of behavior is likely for multistrand rope-like macromolecules where localized separation or partial untwisting of the intertwined chains occurs, creating temporary hinges, in accordance with biochemical evidence for sequence-specific sites of flexibility. Results are presented on the entanglement effects at high concentrations. We have calculated rotational diffusion rates as a function of concentration and molecular weight. Using PFGE to measure the molecular weight profiles, our fracture studies at high strain rates demonstrate chain halving and quartering in accordance with the predictions of the thermally activated barrier to scission theory for single-chain polymers.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

The melting behavior of a DNA junction structure: a calorimetric and spectroscopic study

We present an investigation of the helix–coil transition in a stable branched oligomer of DNA, known as an immobile DNA junction. This junction is composed of four 16-mer strands, which yield four double-helical arms, each containing 8 nucleotide pairs. Properties of the individual arms of this complex are modeled by four octameric duplexes. We have performed experiments using calorimetry, uv absorbance, and CD spectroscopy to characterize the melting transitions of the junction and each arm. By comparing our spectroscopic and calorimetric results on the junction and its component arms, we are able to conclude the following: (1) The calorimetric transition enthalpy for the overall junction complex is equal to the sum of the calorimetric transition enthalpies of the four constituent duplex arms. (2) The optical and the calorimetric measurements yield qualitatively similar, but not identical thermodynamic data. (3) The melting temperature of the junction is less dependent on concentration than the melting temperatures of the individual arms. We attribute this observation to the tetrameric nature of the junction. (4) The ratio of the calorimetric transition enthalpy of the junction and its corresponding van't Hoff value is close to unity. (5) The CD spectrum of the junction is equal quantitatively to the sum of the B-like CD spectra of the four constituent duplex arms.     

MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities.     

Sequence-dependent mechanics of single DNA molecules

Atomic force microscope-based single-molecule force spectroscopy was employed to measure sequence-dependent mechanical properties of DNA by stretching individual DNA double strands attached between a gold surface and an AFM tip. We discovered that in lambda-phage DNA the previously reported B-S transition, where 'S' represents an overstretched conformation, at 65 pN is followed by a nonequilibrium melting transition at 150 pN. During this transition the DNA is split into single strands that fully recombine upon relaxation. The sequence dependence was investigated in comparative studies with poly(dG-dC) and poly(dA-dT) DNA. Both the B-S and the melting transition occur at significantly lower forces in poly(dA-dT) compared to poly(dG-dC). We made use of the melting transition to prepare single poly(dG-dC) and poly(dA-dT) DNA strands that upon relaxation reannealed into hairpins as a result of their self-complementary sequence. The unzipping of these hairpins directly revealed the base pair-unbinding forces for G-C to be 20 +/- 3 pN and for A-T to be 9 +/- 3 pN.     

Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity

N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmol s⁻¹ in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.     

Physical and topological properties of circular DNA

Several types of circular DNA molecules are now known. These are classified as single-stranded rings, covalently closed duplex rings, and weakly bonded duplex rings containing an interruption in one or both strands. Single rings are exemplified by the viral DNA from φX174 bacteriophage. Duplex rings appear to exist in a twisted configuration in neutral salt solutions at room temperature. Examples of such molecules are the DNA''s from the papova group of tumor viruses and certain intracellular forms of φX and λ-DNA. These DNA''s have several common properties which derive from the topological requirement that the winding number in such molecules is invariant. They sediment abnormally rapidly in alkaline (denaturing) solvents because of the topological barrier to unwinding. For the same basic reason these DNA''s are thermodynamically more stable than the strand separable DNA''s in thermal and alkaline melting experiments. The introduction of one single strand scission has a profound effect on the properties of closed circular duplex DNA''s. In neutral solutions a scission appears to generate a swivel in the complementary strand at a site in the helix opposite to the scission. The twists are then released and a slower sedimenting, weakly closed circular duplex is formed. Such circular duplexes exhibit normal melting behavior, and in alkali dissociate to form circular and linear single strands which sediment at different velocities. Weakly closed circular duplexes containing an interruption in each strand are formed by intramolecular cyclization of viral λ-DNA. A third kind of weakly closed circular duplex is formed by reannealing single strands derived from circularly permuted T2 DNA. These reconstituted duplexes again contain an interruption in each strand though not necessarily regularly spaced with respect to each other.     

Polycationic ligands of different chemical classes stimulate DNA strand displacement between short oligonucleotides in a protein‐free system

The ability of polycationic ligands to stimulate DNA strand displacement between short oligonucleotides in a protein‐free system is demonstrated. We show that two ligands, tetracationic aliphatic amine (spermine) and a dicationic intercalating drug (chloroquine), promote strand displacement in a concentration‐dependent manner. At low concentrations both ligands decelerate spontaneous strand displacement because of their impact on the stability of the DNA duplex. At elevated concentrations they accelerate strand displacement via formation of intermediate structures containing three DNA strands. The rate of the last process does not correlate with the thermal dissociation rate of the entire DNA duplex. It indicates that, possibly, the action of these agents cannot be explained by their influence on the stability of the DNA duplex. In general, our results suggest that the ability to stimulate DNA strand displacement appears to be a common feature of polycations of different chemical and structural classes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 633–641, 2016.     

Kinetics of denaturation of DNA

The kinetics of denaturation of DNA have been studied by relaxation techniques. Examination of the terminal relaxation times for a variety of DNA's under a variety of conditions has shown that DNA denaturation is principally a hydrodynamically limited process. Measurements within the helix–coil transition have demonstrated that the experimentally measured terminal relaxation times are a function of the following: (1) position in the helix–coil transition; (2) ionic strength of the solvent; (3) solvent viscosity; (4) DNA concentration; (5) molecular weight; (6) number and position of single-strand breaks. The dependence of the terminal relaxation time on the above mentioned factors can be attributed to hydrodynamic effects. Thus a hydrodynamic model for DNA unwinding is required. The model which best fits the data involves the assumption of a rotational frictional coefficient independent of molecular weight. This assumption is suggested by the fact that the relaxation time is proportional to the first power of the molecular weight.     

Damage by Visible Light to the Acridine Orange-DNA Complex

Salmon DNA has been irradiated with visible light in the presence of acridine orange. If the dye is bound to the DNA, there results: (a) a decrease in sedimentation coefficient, (b) a lowering of viscosity, and (c) a decrease in the thermal denaturation temperature. CsCl banding experiments show that the first two effects reflect depolymerization of the DNA. Depolymerization apparently occurs by single-strand scission although some double-strand scission is not excluded. The destabilization of secondary structure results probably from chemical attack on the components of the individual strands.     

Site-specific interaction of intercalating drugs with a branched DNA molecule

The interaction of a stable branched DNA molecule with an intercalative drug is probed by hydroxyl radical scission. Methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)], consisting of an intercalating ring system tethered to EDTA.Fe(II), produces the hydroxyl radicals by means of a Fenton reaction. The cleavage patterns of each labeled strand in a branched tetramer of four 16-mers are compared with those of the same strands in unbranched duplex controls. Strong differences between the profiles corresponding to scission of branched and duplex DNA molecules are seen in each of the strands at low MPE/DNA ratios. A specific site in the branched structure interacts preferentially with the drug, while other regions of the molecule are protected from cleavage. At 4 degrees C, cutting at strand positions demarcating the site of enhanced affinity is observed to be 60-100% more efficient than at the corresponding sequence positions in the control duplex DNA molecules; the degree of protection is comparable. Cleavage in the vicinity of the preferred site occurs at residues flanking the branch point. The reactive Fe(II) group appears to be centered within two residues of the branch point, and the site of preferential intercalation may be between the two base pairs abutting the branch point in one of the two helical domains. The pattern of preferential cutting at this site is eliminated in the presence of excess propidium diiodide, another intercalative drug.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson-Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37 degrees C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.