9 research outputs found
Lifetimes and Reaction Pathways of Guanine Radical Cations and Neutral Guanine Radicals in an Oligonucleotide in Aqueous Solutions
The exposure of guanine in the oligonucleotide 5′-dÂ(TCGCT)
to one-electron oxidants leads initially to the formation of the guanine
radical cation G<sup>•+</sup>, its deptotonation product GÂ(-H)<sup>•</sup>, and, ultimately, various two- and four-electron oxidation
products via pathways that depend on the oxidants and reaction conditions.
We utilized single or successive multiple laser pulses (308 nm, 1
Hz rate) to generate the oxidants CO<sub>3</sub><sup>•–</sup> and SO<sub>4</sub><sup>•–</sup> (via the photolysis
of S<sub>2</sub>O<sub>8</sub><sup>2–</sup> in aqueous solutions
in the presence and absence of bicarbonate, respectively) at concentrations/pulse
that were ∼20-fold lower than the concentration of 5′-dÂ(TCGCT).
Time-resolved absorption spectroscopy measurements following single-pulse
excitation show that the G<sup>•+</sup> radical (p<i>K</i><sub>a</sub> = 3.9) can be observed only at low pH and is hydrated
within 3 ms at pH 2.5, thus forming the two-electron oxidation
product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single
pulse excitation, the principal reactive intermediate is GÂ(-H)<sup>•</sup>, which, at best, reacts only slowly with H<sub>2</sub>O and lives for ∼70 ms in the absence of oxidants/other radicals
to form base sequence-dependent intrastrand cross-links via the nucleophilic
addition of N3-thymidine to C8-guanine (5′-G*CT* and 5′-T*CG*).
Alternatively, GÂ(-H)<sup>•</sup> can be oxidized further by
reaction with CO<sub>3</sub><sup>•–</sup>, generating
the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin
(2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin
(Gh) and spiroiminodihydantoin (Sp), appear only after a second (or
more) laser pulse. The levels of all products, except 8-oxoG, which
remains at a low constant value, increase with the number of laser
pulses
Water Oxidation Catalyzed by Cobalt(II) Adsorbed on Silica Nanoparticles
A novel, highly efficient, and stable water oxidation
catalyst
was prepared by a pH-controlled adsorption of CoÂ(II) on ∼10
nm diameter silica nanoparticles. A <i>lower limit</i> of
∼300 s<sup>–1</sup> per cobalt atom for the catalyst
turnover frequency in oxygen evolution was estimated, which attests
to a very high catalytic activity. Electron microscopy revealed that
cobalt is adsorbed on the SiO<sub>2</sub> nanoparticle surfaces as
small (1–2 nm) clusters of CoÂ(OH)<sub>2</sub>. This catalyst
is optically transparent over the entire UV–vis range and is
thus suitable for mechanistic investigations by time-resolved spectroscopic
techniques
Mechanistic Aspects of Hydration of Guanine Radical Cations in DNA
The mechanistic aspects of hydration
of guanine radical cations,
G<sup>•+</sup> in double- and single-stranded oligonucleotides
were investigated by direct time-resolved spectroscopic monitoring
methods. The G<sup>•+</sup> radical one-electron oxidation
products were generated by SO<sub>4</sub><sup>•–</sup> radical anions derived from the photolysis of S<sub>2</sub>O<sub>8</sub><sup>2–</sup> anions by 308 nm laser pulses. In neutral
aqueous solutions (pH 7.0), after the complete decay of SO<sub>4</sub><sup>•–</sup> radicals (∼5 μs after the
actinic laser flash) the transient absorbance of neutral guanine radicals,
GÂ(-H)<sup>•</sup> with maximum at 312 nm, is dominant. The
kinetics of decay of GÂ(-H)<sup>•</sup> radicals depend strongly
on the DNA secondary structure. In double-stranded DNA, the GÂ(-H)<sup>•</sup> decay is biphasic with one component decaying with
a lifetime of ∼2.2 ms and the other with a lifetime of ∼0.18
s. By contrast, in single-stranded DNA the GÂ(-H)<sup>•</sup> radicals decay monophasically with a ∼ 0.28 s lifetime. The
ms decay component in double-stranded DNA is correlated with the enhancement
of 8-oxo-7,8-dihydroguanine (8-oxoG) yields which are ∼7 greater
than in single-stranded DNA. In double-stranded DNA, it is proposed
that the GÂ(-H)<sup>•</sup> radicals retain radical cation character
by sharing the N1-proton with the N3-site of C in the [G<sup>•+</sup>:C] base pair. This [GÂ(-H)<sup>•</sup>:H<sup>+</sup>C ⇆
G<sup>•+</sup>:C] equilibrium allows for the hydration of G<sup>•+</sup> followed by formation of 8-oxoG. By contrast, in
single-stranded DNA, deprotonation of G<sup>•+</sup> and the
irreversible escape of the proton into the aqueous phase competes
more effectively with the hydration mechanism, thus diminishing the
yield of 8-oxoG, as observed experimentally
Thermodynamic Profiles and Nuclear Magnetic Resonance Studies of Oligonucleotide Duplexes Containing Single Diastereomeric Spiroiminodihydantoin Lesions
The spiroiminodihydantoins (Sp) are highly mutagenic
oxidation
products of guanine and 8-oxo-7,8-dihydroguanine in DNA. The Sp lesions
have recently been detected in the liver and colon of mice infected
with <i>Helicobacter hepaticus</i> that induces inflammation
and the development of liver and colon cancers in murine model systems
[Mangerich, A., et al. (2012) <i>Proc. Natl. Acad. Sci. U.S.A.
109</i>, E1820–E1829]. The impact of Sp lesions on the
thermodynamic characteristics and the effects of the diastereomeric
Sp-<i>R</i> and Sp-<i>S</i> lesions on the conformational
features of double-stranded 11-mer oligonucleotide duplexes have been
studied by a combination of microcalorimetric methods, analysis of
DNA melting curves, and two-dimensional nuclear magnetic resonance
methods. The nonplanar, propeller-like shapes of the Sp residues strongly
diminish the extent of local base stacking interactions that destabilize
the DNA duplexes characterized by unfavorable enthalpy contributions.
Relative to that of an unmodified duplex, the thermally induced unfolding
of the duplexes with centrally positioned Sp-<i>R</i> and
Sp-<i>S</i> lesions into single strands is accompanied by
a smaller release of cationic counterions (Δ<i>n</i><sub>Na<sup>+</sup></sub> = 0.6 mol of Na<sup>+</sup>/mol of duplex)
and water molecules (Δ<i>n</i><sub>w</sub> = 17 mol
of H<sub>2</sub>O/mol of duplex). The unfolding parameters are similar
for the Sp-<i>R</i> and Sp-<i>S</i> lesions, although
their orientations in the duplexes are different. The structural disturbances
radiate one base pair beyond the flanking C:G pair, although Watson–Crick
hydrogen bonding is maintained at all flanking base pairs. The observed
relatively strong destabilization of B-form DNA by the physically
small Sp lesions is expected to have a significant impact on the processing
of these lesions in biological environments
The Nonbulky DNA Lesions Spiroiminodihydantoin and 5‑Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation <i>in Vitro</i>
The
most common, oxidatively generated lesion in cellular DNA is
8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly
mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh)
in DNA. In human cell-free extracts, both lesions can be excised by
base excision repair and global genomic nucleotide excision repair.
However, it is not known if these lesions can be removed by transcription-coupled
DNA repair (TCR), a pathway that clears lesions from DNA that impede
RNA synthesis. To determine if Sp or Gh impedes transcription, which
could make each a viable substrate for TCR, either an Sp or a Gh lesion
was positioned on the transcribed strand of DNA under the control
of a promoter that supports transcription by human RNA polymerase
II. These constructs were incubated in HeLa nuclear extracts that
contained active RNA polymerase II, and the resulting transcripts
were resolved by denaturing polyacrylamide gel electrophoresis. The
structurally rigid Sp strongly blocks transcription elongation, permitting
1.6 ± 0.5% nominal lesion bypass. In contrast, the conformationally
flexible Gh poses less of a block to human RNAPII, allowing 9 ±
2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is
minimally affected by glycosylase activity found in the HeLa nuclear
extract. These data specifically suggest that both Sp and Gh may well
be susceptible to TCR because each poses a significant block to human
RNA polymerase II progression. A more general principle is also proposed:
Conformational flexibility may be an important structural feature
of DNA lesions that enhances their transcriptional bypass
Differences in the Access of Lesions to the Nucleotide Excision Repair Machinery in Nucleosomes
In
nucleosomes, the access of DNA lesions to nucleotide excision
repair is hindered by histone proteins. However, evidence that the
nature of the DNA lesions may play a role in facilitating access is
emerging, but these phenomena are not well-understood. We have used
molecular dynamics simulations to elucidate the structural, dynamic,
and energetic properties of the <i>R</i> and <i>S</i> 5′-8-cyclo-2′-dG and the (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG lesions in a nucleosome. Our results show
that the (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG adduct
is more dynamic and more destabilizing than the smaller and more constrained
5′,8-cyclo-2′-dG lesions, suggesting more facile access
to the more bulky (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG lesion
Generation of Guanine–Thymine Cross-Links in Human Cells by One-Electron Oxidation Mechanisms
The
one-electron oxidation of cellular DNA in cultured human HeLa
cells initiated by intense nanosecond 266 nm laser pulse irradiation
produces cross-links between guanine and thymine bases (G*-T*), characterized
by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms.
The DNA lesions were quantified by isotope dilution LC-MS/MS methods
in the multiple reaction-monitoring mode using isotopically labeled
[<sup>15</sup>N, <sup>13</sup>C]-nucleotides as internal standards.
Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions,
the G*-T* cross-linked lesions were detected at levels of ∼0.21
and 1.19 dÂ(G*-T*) lesions per 10<sup>6</sup> DNA bases at laser intensities
of 50 and 280 mJ/cm<sup>2</sup>/pulse, respectively
Differences in the Access of Lesions to the Nucleotide Excision Repair Machinery in Nucleosomes
In
nucleosomes, the access of DNA lesions to nucleotide excision
repair is hindered by histone proteins. However, evidence that the
nature of the DNA lesions may play a role in facilitating access is
emerging, but these phenomena are not well-understood. We have used
molecular dynamics simulations to elucidate the structural, dynamic,
and energetic properties of the <i>R</i> and <i>S</i> 5′-8-cyclo-2′-dG and the (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG lesions in a nucleosome. Our results show
that the (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG adduct
is more dynamic and more destabilizing than the smaller and more constrained
5′,8-cyclo-2′-dG lesions, suggesting more facile access
to the more bulky (+)-<i>cis-anti</i>-BÂ[<i>a</i>]ÂP-dG lesion
Sequence-Dependent Variation in the Reactivity of 8-Oxo-7,8-dihydro-2′-deoxyguanosine toward Oxidation
The goal of this study was to define the effect of DNA
sequence
on the reactivity of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG)
toward oxidation. To this end, we developed a quadrupole/time-of-flight
(QTOF) mass spectrometric method to quantify the reactivity of site
specifically modified oligodeoxyribonucleotides with two model oxidants:
nitrosoperoxycarbonate (ONOOCO<sub>2</sub><sup>–</sup>), a
chemical mediator of inflammation, and photoactivated riboflavin,
a classical one-electron oxidant widely studied in mutagenesis and
charge transport in DNA. In contrast to previous observations with
guanine [Margolin, Y., (2006) Nat. Chem. Biol. 2, 365], sequence context
did not affect the reactivity of ONOOCO<sub>2</sub><sup>–</sup> with 8-oxodG, but photosensitized riboflavin showed a strong sequence
preference in its reactivity with the following order (8-oxodG = O):
COA ≈ AOG > GOG ≥ COT > TOC > AOC. That the
COA context
was the most reactive was unexpected and suggests a new sequence context
where mutation hotspots might occur. These results point to both sequence-
and agent-specific effects on 8-oxodG oxidation