Copper-dependent DNA damage induced by hydrazobenzene, an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2'-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Synthesis of Photo-Responsive Acridine-Modified DNA and Its Application to Site-Selective RNA Scission

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the T_m values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission.     

Synthesis of photo-responsive acridine-modified DNA and its application to site-selective RNA scission

Photo-responsive phosphoramidite monomers, which bear an azobenzene between acridine and the phosphoramidite unit, were synthesized, and incorporated into oligonucleotides. Upon UV irradiation, the azobenzene in the modified DNA efficiently isomerized from the trans isomer into the cis isomer. Although the T(m) values of their duplexes with complementary DNA were not much changed by the isomerization, site-selective RNA scission was significantly accelerated by the UV irradiation when Mn(II) ion was used as the catalyst for RNA scission.     

Conformational changes of DNA by photoirradiation of DNA-bis(Zn(II)-cyclen)-azobenzene complex

Bis(Zn(II)-cyclen)-azobenzene derivative, which has two Zn(II)-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent fordoublestranded DNA in aqueous solution. The Zn(II)-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn(II)-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn(II)-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn(II)-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner     

Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Using azobenzene incorporated DNA aptamers to probe molecular binding interactions

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.     

Photo-regulated duplex- and triplex-formation of the modified DNA carrying azobenzene.

The duplex- and triplex- forming activity of oligonucleotide was photo-regulated by using the isomerization of azobenzene in the side chain. When the azobenzene was isomerized from the trans-form to the cis-form by photo-irradiation, the melting temperatures of the duplex and triplex between the oligonucleotide and its complementary counterpart were significantly lowered.     

Azobenzene Photoisomerization-Induced Destabilization of B-DNA

Molecular photoswitches provide a promising way for selective regulation of nanoscaled biological systems. It has been shown that conformational changes of azobenzene, one of the widely used photoswitches, can be used to reversibly control DNA duplex formation. Here, we investigate the conformational response of DNA upon azobenzene binding and isomerization, using a threoninol linker that has been experimentally investigated recently. To this end, nonequilibrium molecular dynamics simulations are carried out using a switching potential describing the photoinduced isomerization. Attachment of azobenzene leads to a distortion of the DNA helical conformation that is similar for the trans and cis forms. However, the trans form is stabilized by favorable stacking interactions whereas the cis form is found to remain flipped out of the basepair-stacked position. Multiple azobenzene attachment augments the distortion in DNA helical conformation. The distorted DNA retains nativelike pairing of bases at ambient temperatures, but shows weaker basepairing compared to native DNA at an elevated temperature.     

Photomodulation of PS-modified oligonucleotides containing azobenzene substituent at pre-selected positions in phosphate backbone

A new protocol has been developed for incorporation of a photoisomerizable azobenzene moiety into synthetic stereo-enriched [R_p] and [S_p] PS-oligonucleotides. The azobenzene pendant is attached at pre-selected positions in internucleotidic phosphorothioate oligonucleotides of both [R_p] and [S_p] diastereomers using a novel reagent, N-iodoacetyl-p-aminoazobenzene, 1. The modified oligomers are purified on HPLC, characterized by LC–MS, and examined for their thermal and photoisomerization properties. The azobenzene moiety imparts greater stability to oligomer duplexes in (E) NN configuration as compared to (Z) configuration. The placement of the azobenzene pendant close to 5′-terminus (n − 1) and 3′-terminus of the modified PS-oligos contributes maximum stability to the duplex while a gradual decline in stability occurs with azobenzene moving toward middle of the duplex. Circular Dichroism studies reveal that the chiral environment at the phosphorus center of the PS-oligos does not alter the global conformation of the DNA duplex as such, suggesting conservation of conformation of the modified DNA strands.     

Design, synthesis, and DNA binding properties of photoisomerizable azobenzene-distamycin conjugates: an experimental and computational study

Here, we present the synthesis, photochemical, and DNA binding properties of three photoisomerizable azobenzene-distamycin conjugates in which two distamycin units were linked via electron-rich alkoxy or electron-withdrawing carboxamido moieties with the azobenzene core. Like parent distamycin A, these molecules also demonstrated AT-specific DNA binding. Duplex DNA binding abilities of these conjugates were found to depend upon the nature and length of the spacer, the location of protonatable residues, and the isomeric state of the conjugate. The changes in the duplex DNA binding efficiency of the individual conjugates in the dark and with their respective photoirradiated forms were examined by circular dichroism, thermal denaturation of DNA, and Hoechst displacement assay with poly[d(A-T).d(T-A)] DNA in 150 mM NaCl buffer. Computational structural analyses of the uncomplexed ligands using ab initio HF and MP2 theory and molecular docking studies involving the conjugates with duplex d[(GC(AT)10CG)]2 DNA were performed to rationalize the nature of binding of these conjugates.     

Conformational and phase transitions in DNA—photosensitive surfactant solutions: Experiment and modeling

DNA binding to trans‐ and cis‐isomers of azobenzene containing cationic surfactant in 5 mM NaCl solution was investigated by the methods of dynamic light scattering (DLS), low‐gradient viscometry (LGV), atomic force microscopy (AFM), circular dichroism (CD), gel electrophoresis (GE), flow birefringence (FB), UV–Vis spectrophotometry. Light‐responsive conformational transitions of DNA in complex with photosensitive surfactant, changes in DNA optical anisotropy and persistent length, phase transition of DNA into nanoparticles induced by high surfactant concentration, as well as transformation of surfactant conformation under its binding to macromolecule were studied. Computer simulations of micelles formation for cis‐ and trans‐isomers of azobenzene containing surfactant, as well as DNA‐surfactant interaction, were carried out. Phase diagram for DNA‐surfactant solutions was designed. The possibility to reverse the DNA packaging induced by surfactant binding with the dilution and light irradiation was shown. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 109–122, 2015.     

Reversible photocontrol of DNA binding by a designed GCN4-bZIP protein

Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.     

Restriction endonuclease SsoII with photoregulated activity--a "molecular gate" approach

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular gate" approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R.SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.     

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA.     

Gold nanoparticles used as a carrier enhance production of anti-hapten IgG in rabbit: a study with azobenzene-dye as a hapten presented on the entire surface of gold nanoparticles

The azobenzene moiety, well-known not only for its reversible cis-to-trans photoisomerization but also as a hapten, does not induce antibodies on its own, but it reacts with antibodies raised against conjugates with protein carriers. Hence we selected azobenzene dye as an indicator to assess the possibility of having gold nano-particles act as an immunological carrier instead of protein carriers. In rabbits, we confirmed an in vivo response against azobenzene dye presented on the entire surface of gold nanoparticles (azo-nanoparticles), where the gold nanoparticles appeared to play a role as a carrier for the hapten. A high yield of immunoglobulin G (IgG) against the azobenzene derivative took place in rabbits injected with azo-nanoparticles, whereas no increase in IgG was recognized in other rabbits treated solely with chemically equivalent azobenzene dye instead of azo-nanoparticles. Electron microscopy and surface plasmon resonance spectroscopy indicated that the IgG obtained specifically recognized the difference between the isomer conformations of the azobenzene moiety.     

DNA synthesis on discontinuous templates by human DNA polymerases: implications for non-homologous DNA recombination.

DNA polymerases catalyze the synthesis of DNA using a continuous uninterrupted template strand. However, it has been shown that a 3'-->5' exonuclease-deficient form of the Klenow fragment of Escherichia coli DNA polymerase I as well as DNA polymerase of Thermus aquaticus can synthesize DNA across two unlinked DNA templates. In this study, we used an oligonucleotide-based assay to show that discontinuous DNA synthesis was present in HeLa cell extracts. DNA synthesis inhibitor studies as well as fractionation of the extracts revealed that most of the discontinuous DNA synthesis was attributable to DNA polymerase alpha. Additionally, discontinuous DNA synthesis could be eliminated by incubation with an antibody that specifically neutralized DNA polymerase alpha activity. To test the relative efficiency of each nuclear DNA polymerase for discontinuous synthesis, equal amounts (as measured by DNA polymerase activity) of DNA polymerases alpha, beta, delta (+/- PCNA) and straightepsilon (+/- PCNA) were used in the discontinuous DNA synthesis assay. DNA polymerase alpha showed the most discontinuous DNA synthesis activity, although small but detectable levels were seen for DNA polymerases delta (+PCNA) and straightepsilon (- PCNA). Klenow fragment and DNA polymerase beta showed no discontinuous DNA synthesis, although at much higher amounts of each enzyme, discontinuous synthesis was seen for both. Discontinuous DNA synthesis by DNA polymerase alpha was seen with substrates containing 3 and 4 bp single-strand stretches of complementarity; however, little synthesis was seen with blunt substrates or with 1 bp stretches. The products formed from these experiments are structurally similar to that seen in vivo for non-homologous end joining in eukaryotic cells. These data suggest that DNA polymerase alpha may be able to rejoin double-strand breaks in vivo during replication.     

Synthesis of oligonucleotides containing a new azobenzene fragment with efficient photoisomerizability

The azobenzene derivatives possessing substituents of ROCH₂CH₂O- and-CH₂CH₂OR′ or -CONHCH₂CH₂OR′ at p,p′-positions, where R and R′ are 4,4′-dimethoxytrityl and 2-cyanoethyl-N,N′-diisopropylphophoramidite, have been synthesized for linking two oligonucelotide segments. It has been found that the azobenzene linkers efficiently undergo trans–cis isomerization by exposing to UV light. The conversion efficiency showed slight dependence on structure or conformation of oligonucleotides attached to the azobenzene chromophore. The cis-form of the azobenzene in oligonucleotides was sufficiently stable at low temperature under dark. The present findings would open the way for light switch of nucleic acid structures.     

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.