32 research outputs found
GâQuadruplex Folds of the Human Telomere Sequence Alter the Site Reactivity and Reaction Pathway of Guanine Oxidation Compared to Duplex DNA
Telomere
shortening occurs during oxidative and inflammatory stress
with guanine (G) as the major site of damage. In this work, a comprehensive
profile of the sites of oxidation and structures of products observed
from G-quadruplex and duplex structures of the human telomere sequence
was studied in the G-quadruplex folds (hybrid (K<sup>+</sup>), basket
(Na<sup>+</sup>), and propeller (K<sup>+</sup> + 50% CH<sub>3</sub>CN)) resulting from the sequence 5â˛-(TAGGGT)<sub>4</sub>T-3â˛
and in an appropriate duplex containing one telomere repeat. Oxidations
with four oxidant systems consisting of riboflavin photosensitization,
carbonate radical generation, singlet oxygen, and the copper Fenton-like
reaction were analyzed under conditions of low product conversion
to determine relative reactivity. The one-electron oxidants damaged
the 5â˛-G in G-quadruplexes leading to spiroiminodihydantoin
(Sp) and 2,2,4-triamino-2<i>H</i>-oxazol-5-one (Z) as major
products as well as 8-oxo-7,8-dihydroguanine (OG) and 5-guanidinohydantoin
(Gh) in low relative yields, while oxidation in the duplex context
produced damage at the 5â˛- and middle-Gs of GGG sequences and
resulted in Gh being the major product. Addition of the reductant <i>N</i>-acetylcysteine (NAC) to the reaction did not alter the
riboflavin-mediated damage sites but decreased Z by 2-fold and increased
OG by 5-fold, while not altering the hydantoin ratio. However, NAC
completely quenched the CO<sub>3</sub><sup>â˘â</sup> reactions.
Singlet oxygen oxidations of the G-quadruplex showed reactivity at
all Gs on the exterior faces of G-quartets and furnished the product
Sp, while no oxidation was observed in the duplex context under these
conditions, and addition of NAC had no effect. Because a long telomere
sequence would have higher-order structures of G-quadruplexes, studies
were also conducted with 5â˛-(TAGGGT)<sub>8</sub>-T-3â˛,
and it provided oxidation profiles similar to those of the single
G-quadruplex. Lastly, Cu<sup>II</sup>/H<sub>2</sub>O<sub>2</sub>-mediated
oxidations were found to be indiscriminate in the damage patterns,
and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) was found to
be a major duplex product, while nearly equal yields of 2Ih and Sp
were observed in G-quadruplex contexts. These findings indicate that
the nature of the secondary structure of folded DNA greatly alters
both the reactivity of G toward oxidative stress as well as the product
outcome and suggest that recognition of damage in telomeric sequences
by repair enzymes may be profoundly different from that of B-form
duplex DNA
Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8âOxo-7,8-dihydroguanine and A and G opposite 5âGuanidinohydantoin and Spiroiminodihydantoin Diastereomers
Reactive oxygen species,
both endogenous and exogenous, can damage
nucleobases of RNA and DNA. Among the nucleobases, guanine has the
lowest redox potential, making it a major target of oxidation. Although
RNA is more prone to oxidation than DNA is, oxidation of guanine in
RNA has been studied to a significantly lesser extent. One of the
reasons for this is that many tools that were previously developed
to study oxidation of DNA cannot be used on RNA. In the study presented
here, the lack of a method for seeking sites of modification in RNA
where oxidation occurs is addressed. For this purpose, reverse transcription
of RNA containing major products of guanine oxidation was used. Extension
of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine
(OG), 5-guanidinohydantoin (Gh), or the <i>R</i> and <i>S</i> diastereomers of spiroiminodihydantoin (Sp) was studied
under standing start conditions. SuperScript III reverse transcriptase
is capable of bypassing these lesions in RNA inserting predominantly
A opposite OG, predominantly G opposite Gh, and almost an equal mixture
of A and G opposite the Sp diastereomers. These data should allow
RNA sequencing of guanine oxidation products by following characteristic
mutation signatures formed by the reverse transcriptase during primer
elongation past G oxidation sites in the template RNA strand
Interactions of the Human Telomere Sequence with the Nanocavity of the ÎąâHemolysin Ion Channel Reveal Structure-Dependent Electrical Signatures for Hybrid Folds
Human
telomeric DNA consists of tandem repeats of the sequence
5â˛-TTAGGG-3â˛, including a 3Ⲡterminal single-stranded
overhang of 100â200 nucleotides that can fold into quadruplex
structures in the presence of suitable metal ions. In the presence
of an applied voltage, the Îą-hemolysin (Îą-HL) protein
ion channel can produce unique current patterns that are found to
be characteristic for various interactions between G-quadruplexes
and the protein nanocavity. In this study, the human telomere in a
complete sequence context, 5â˛-TAGGGÂ(TTAGGG)<sub>3</sub>TT-3â˛,
was evaluated with respect to its multiple folding topologies. Notably,
the coexistence of two interchangeable conformations of the K<sup>+</sup>-induced folds, hybrid-1 and hybrid-2, were readily resolved
at a single-molecule level along with triplex folding intermediates,
whose characterization has been challenging in experiments that measure
the bulk solution. These results enabled us to profile the thermal
denaturation process of these structures to elucidate the relative
distributions of hybrid-1, hybrid-2, and folding intermediates such
as triplexes. For example, at 37 °C, pH 7.9, in 50 mM aqueous
KCl, the ratio of hybrid-1:hybrid-2:triplex is approximately 11:5:1
in dilute solution. The results obtained lay the foundation for utilizing
the Îą-HL ion channel as a simple tool for monitoring how small
molecules and physical context shift the equilibrium between the many
G-quadruplex folds of the human telomere sequence
Sequencing the Mouse Genome for the Oxidatively Modified Base 8âOxo-7,8-dihydroguanine by OG-Seq
Oxidative
damage to the genome can yield the base 8-oxo-7,8-dihydroguanine
(OG). In vitro studies suggested OG would preferentially form in 5â˛-GG-3â˛
sequence contexts after exposure to reactive oxygen species. Herein,
OG locations in the genome were studied by development of âOG-Seqâ
to sequence OG sites via next-generation sequencing at âź0.15-kb
resolution. The results of this study found âź10âŻ000
regions of OG enrichment in WT mouse embryonic fibroblasts and âź18âŻ000
regions when the OG repair glycosylase Ogg1 was knocked out. Gene
promoters and UTRs harbor more OG-enriched sites than expected if
the sites were randomly distributed throughout the genome and correlate
with reactive 5â˛-GG-3Ⲡsequences, a result supporting
decades of in vitro studies. Sequencing of OG paves the way to address
chemical and biological questions surrounding this modified DNA base,
such as its role in disease-specific mutations and its epigenetic
potential in gene regulation
Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps
Modifications
to nucleotides in the genome can lead to mutations
or are involved in regulation of gene expression, and therefore, finding
the site of modification is a worthy goal. Robust methods for sequencing
modification sites on commercial sequencers have not been developed
beyond the epigenetic marks on cytosine. Herein, a method to sequence
DNA modification sites was developed that utilizes DNA glycosylases
found in the base excision repair pathway to excise the modification.
This approach yields a gap at the modification site that is sealed
by T4-DNA ligase, yielding a product strand missing the modification.
Upon sequencing, the modified nucleotide is reported as a deletion
mutation, identifying its location. This approach was used to detect
a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the <i>KRAS</i> gene in synthetic oligoÂdeoxyÂnucleotides.
Additionally, an OG modification site was placed in the <i>VEGF</i> promoter in a plasmid and sequenced. This method requires only commercially
available materials and can be put into practice on any sequencing
platform, allowing this method to have broad potential for finding
modifications in DNA
Human Telomere GâQuadruplexes with Five Repeats Accommodate 8âOxo-7,8-dihydroguanine by Looping out the DNA Damage
Inflammation
and oxidative stress generate free radicals that oxidize
guanine (G) in DNA to 8-oxo-7,8-dihydroguanine (OG), and this reaction
is prominent in the G-rich telomere sequence. In telomeres, OG is
not efficiently removed by repair pathways allowing its concentration
to build, surprisingly without any immediate negative consequences
to stability. Herein, OG was synthesized in five repeats of the human
telomere sequence (TTAGGG)<sub><i>n</i></sub>, at the 5â˛-G
of the 5â˛-most, middle, and 3â˛-most G tracks, representing
hotspots for oxidation. These synthetic oligomers were folded in relevant
amounts of K<sup>+</sup>/Na<sup>+</sup> to adopt hybrid G-quadruplex
folds. The structural impact of OG was assayed by circular dichroism,
thermal melting, <sup>1</sup>H NMR, and single-molecule profiling
by the Îą-hemolysin nanopore. On the basis of these results,
OG was well accommodated in the five-repeat sequences by looping out
the damaged G track to allow the other four tracks to adopt a hybrid
G-quadruplex. These results run counter to previous studies with OG
in four-repeat telomere sequences that found OG to be highly destabilizing
and causing significant reorientation of the fold. When taking a wider
view of the human telomere sequence and considering additional repeats,
we found OG to cause minimal impact on the structure. The plasticity
of this repeat sequence addresses how OG concentrations can increase
in telomeres without immediate telomere instability or attrition
The Fifth Domain in the GâQuadruplex-Forming Sequence of the Human <i>NEIL3</i> Promoter Locks DNA Folding in Response to Oxidative Damage
DNA oxidation is
an inevitable and usually detrimental process,
but the cell is capable of reversing this state because the cell possesses
a highly developed set of DNA repair machineries, including the DNA
glycosylase NEIL3 that is encoded by the <i>NEIL3</i> gene.
In this work, the G-rich promoter region of the human <i>NEIL3</i> gene was shown to fold into a dynamic G-quadruplex (G4) structure
under nearly physiological conditions using spectroscopic techniques
(e.g., nuclear magnetic resonance, circular dichroism, fluorescence,
and ultravioletâvisible) and DNA polymerase stop assays. The
presence of 8-oxo-7,8-dihydroguanine (OG) modified the properties
of the <i>NEIL3</i> G4 and entailed the recruitment of the
fifth domain to function as a âspare tireâ, in which
an undamaged fifth G-track is swapped for the damaged section of the
G4. The polymerase stop assay findings also revealed that owing to
its dynamic polymorphism, the <i>NEIL3</i> G4 is more readily
bypassed by DNA polymerase I (Klenow fragment) than well-known oncogene
G4s are. This study identifies the <i>NEIL3</i> promoter
possessing a G-rich element that can adopt a G4 fold, and when OG
is incorporated, the sequence can lock into a more stable G4 fold
via recruitment of the fifth track of Gs
A Role for the Fifth GâTrack in GâQuadruplex Forming Oncogene Promoter Sequences during Oxidative Stress: Do These âSpare Tiresâ Have an Evolved Function?
Uncontrolled
inflammation or oxidative stress generates electron-deficient
species that oxidize the genome increasing its instability in cancer.
The G-quadruplex (G4) sequences regulating the <i>c-MYC</i>, <i>KRAS</i>, <i>VEGF</i>, <i>BCL-2</i>, <i>HIF-1Îą</i>, and <i>RET</i> oncogenes,
as examples, are targets for oxidation at loop and 5â˛-core
guanines (G) as showcased in this study by CO<sub>3</sub><sup>â˘â</sup> oxidation of the <i>VEGF</i> G4. Products observed include
8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin
(Gh). Our previous studies found that OG and Gh, when present in the
four G-tracks of the solved structure for <i>VEGF</i> and <i>c-MY</i>C, were not substrates for the base excision repair
(BER) DNA glycosylases in biologically relevant KCl solutions. We
now hypothesize that a fifth G-track found a few nucleotides distant
from the G4 tracks involved in folding can act as a âspare
tire,â facilitating extrusion of a damaged G-run into a large
loop that then becomes a substrate for BER. Thermodynamic, spectroscopic,
and DMS footprinting studies verified the fifth domain replacing a
damaged G-track with OG or Gh at a loop or core position in the <i>VEGF</i> G4. These new âspare tireâ-containing
strands with Gh in loops are now found to be substrates for initiation
of BER with the NEIL1, NEIL2, and NEIL3 DNA glycosylases. The results
support a hypothesis in which regulatory G4s carry a âspare-tireâ
fifth G-track for aiding in the repair process when these sequences
are damaged by radical oxygen species, a feature observed in a large
number of these sequences. Furthermore, formation and repair of oxidized
bases in promoter regions may constitute an additional example of
epigenetic modification, in this case of guanine bases, to regulate
gene expression in which the G4 sequences act as sensors of oxidative
stress
Zika Virus Genomic RNA Possesses Conserved GâQuadruplexes Characteristic of the Flaviviridae Family
Zika virus has emerged
as a global concern because neither a vaccine nor antiviral compounds
targeting it exist. A structure for the positive-sense RNA genome
has not been established, leading us to look for potential G-quadruplex
sequences (PQS) in the genome. The analysis identified >60 PQSs
in the Zika genome. To minimize the PQS population, conserved sequences
in the Flaviviridae family were found by sequence alignment, identifying
seven PQSs in the prM, E, NS1, NS3, and NS5 genes. Next, alignment
of 78 Zika strain genomes identified a unique PQS near the end of
the 3â˛-UTR. Structural studies on the G-quadruplex sequences
found four of the conserved Zika virus sequences to adopt stable,
parallel-stranded folds that bind a G-quadruplex-specific compound,
and one that was studied caused polymerase stalling when folded to
a G-quadruplex. Targeting these PQSs with G-quadruplex binding molecules
validated in previous clinical trials may represent a new approach
for inhibiting viral replication
Unfolding Kinetics of the Human Telomere iâMotif Under a 10 pN Force Imposed by the ÎąâHemolysin Nanopore Identify Transient Folded-State Lifetimes at Physiological pH
Cytosine
(C)-rich DNA can adopt i-motif folds under acidic conditions,
with the human telomere i-motif providing a well-studied example.
The dimensions of this i-motif are appropriate for capture in the
nanocavity of the Îą-hemolysin (Îą-HL) protein pore under
an electrophoretic force. Interrogation of the current vs time (<i>i</i>â<i>t</i>) traces when the i-motif interacts
with Îą-HL identified characteristic signals that were pH dependent.
These features were evaluated from pH 5.0 to 7.2, a region surrounding
the transition pH of the i-motif (6.1). When the i-motif without polynucleotide
tails was studied at pH 5.0, the folded structure entered the nanocavity
of Îą-HL from either the top or bottom face to yield characteristic
current patterns. Addition of a 5Ⲡ25-mer poly-2â˛-deoxyadensosine
tail allowed capture of the i-motif from the unfolded terminus, and
this was used to analyze the pH dependency of unfolding. At pH values
below the transition point, only folded strands were observed, and
when the pH was increased above the transition pH, the number of folded
events decreased, while the unfolded events increased. At pH 6.8 and
7.2 4% and 2% of the strands were still folded, respectively. The
lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms,
respectively, at 160 mV electrophoretic force. These lifetimes are
sufficiently long to affect enzymes operating on DNA. Furthermore,
these transient lifetimes are readily obtained using the Îą-HL
nanopore, a feature that is not easily achievable by other methods