of 31
Redox Signaling through DNA
Elizabeth O’Brien
,
Rebekah M.B. Silva
, and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA
91125
Abstract
Biological electron transfer reactions between metal cofactors are critical to many essential
processes within the cell. Duplex DNA is, moreover, capable of mediating the transport of charge
through its
π
-stacked nitrogenous bases. Increasingly, [4Fe4S] clusters, generally redox-active
cofactors, have been found to be associated with enzymes involved in DNA processing. DNA-
binding enzymes containing [4Fe4S] clusters can thus utilize DNA charge transport (DNA CT) for
redox signaling to coordinate reactions over long molecular distances. In particular, DNA CT
signaling may represent the first step in the search for DNA lesions by proteins containing [4Fe4S]
clusters that are involved in DNA repair. Here we describe research carried out to examine the
chemical characteristics and biological consequences of DNA CT. We are finding that DNA CT
among metalloproteins represents powerful chemistry for redox signaling at long range within the
cell.
Graphical Abstract
1. Introduction
Electron transfer reactions are integral components of fundamental processes in biology.
These reactions often occur between two proteins that coordinate metals
[
1
]
such as Fe, Mn,
Zn, or Cu. Metal centers in proteins can facilitate the single electron redox reactions central
to biological processes such as respiration
[
2
]
and photosynthesis
[
3
]
. The mitochondrial
respiratory chain, for example, involves several electron transfer processes, mediated by the
metal cofactors in large, multiprotein complexes.
[
4
12
]
Several iron-sulfur centers and heme
*
to whom correspondence should be addressed at jkbarton@caltech.edu.
HHS Public Access
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Published in final edited form as:
Isr J Chem
. 2016 October ; 56(9-10): 705–723. doi:10.1002/ijch.201600022.
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proteins mediate redox activity essential to driving the oxidative phosphorylation that
aerobic organisms use to generate energy.
[
2
,
4
6
]
These redox reactions and others occur between donors and acceptors that are in relative
proximity to one another; the electron transfer steps driving respiration occur within a single
complex or between adjacent respiratory chain complexes in the mitochondrial
membrane.
[
2
,
4
12
]
As established in beautiful studies by Gray and coworkers, biological
electron transfer reactions can span over distances exceeding the single-step tunneling limit
of ~20Å;
[
4
]
extensive studies with Ru- and Re-modified proteins
[
4
,
13
16
]
directly
demonstrate intraprotein charge transfer over distances greater than this limit. Here,
metalloproteins coordinate these reactions in multiple steps, using multistep electron
tunneling, or electron hopping, through the insulating protein matrix. A strategically
positioned aromatic residue such as tyrosine or tryptophan, with a relatively low ionization
potential,
[
17
,
18
]
can increase the rate of electron transfer through a protein matrix.
[
14
,
16
]
A
mutant azurin containing a covalent [Re(CO)
3
(dmp)]
+
(dmp= 4,7-dimethyl-1,10-
phenantroline) photooxidant coordinated by His 124 and a Trp residue engineered
approximately halfway between the Cu (I) electron donor and photoexcited Re (II) acceptor,
for example, has been demonstrated to transfer charge over a ~20Å distance approximately
100 times faster than the predicted time for the single-step tunneling reaction.
[
16
]
Nature can
thus design proteins optimized for efficient redox chemistry within a macromolecule or a
complex, but can biological electron donors and acceptors also couple with one another from
still longer distances, to coordinate a genome-wide process such as DNA repair?
Here we focus on electron transfer reactions across DNA, another important biological
macromolecule. We describe the extensive characterization of this chemistry in many
laboratories and how study of long-range electron transfer through DNA led us also to
examine the redox chemistry of DNA processing enzymes that coordinate iron cofactors.
The ubiquity of [4Fe4S] clusters being associated with enzymes involved in DNA
processing has led us to consider roles where electron transfer reactions through DNA might
be beneficially applied. This review is not exhaustive, but it is intended to illustrate the
unique features of DNA charge transport chemistry and how these features may be utilized
within the cell.
2. Fundamentals of DNA Charge Transport
Electron transfer through protein matrices occurs on the microsecond timescale,
[
4
]
mediated
by
σ
bonds and hydrogen bonds. Electron transfer reactions occur through duplex DNA,
however, at a significantly faster rate.
[
19
27
]
DNA-mediated charge transport (DNA CT) is
mediated by the
π
-stacking interactions of the nitrogenous bases at the center of the DNA
helix and occurs on the picosecond timescale.
[
27
]
The aromatic bases, stacked in 3.4 Å
layers, contain overlapping
π
orbitals in a structure closely resembling that of stacked
graphene sheets (Figure 1). After observing this similarity in structure between DNA and
graphite in the dry, solid state, Eley and Spivey
[
28
]
along with others predicted that the DNA
helix, based upon its chemical structure, would conduct charge. Our laboratory has observed
that DNA-mediated charge transport occurs over long range
[
22
,
26
]
and is shallowly
dependent on molecular distance traveled.
[
23
,
26
,
29
31
]
DNA CT indeed occurs through the
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nitrogenous base pairs, whose overlapping
π
orbitals provide a pathway for charge to travel
down the helical base pair stack.
[
19
31
]
This property is additionally exquisitely sensitive to
perturbations in the stacking interactions of the bases, such as those that occur with base-pair
mismatches and lesions.
[
23
,
29
31
]
Studies of DNA CT by our laboratory and others have prompted several proposals about the
mechanism through which CT occurs under aqueous, biologically relevant
conditions.
[
23
,
25
39
]
Superexchange between DNA bases was proposed but cannot explain
the long molecular distances over which DNA-mediated CT is observed.
[
32
35
,
40
]
Localized
hopping has also been suggested to mediate DNA CT, as charge can migrate along DNA
through superexchange between neighboring guanine bases in the sequence.
[
41
]
This model,
however, cannot fully explain the weak distance dependence of DNA CT consistently
demonstrated
[
42
44
]
or the attenuation of DNA CT in the presence of base-pair
mismatches.
[
23
,
26
,
29
31
]
Delocalization of charge likely occurs through transient interactions
influenced by dynamic changes in the duplex DNA structure in solution
[
27
,
38
,
45
]
and perhaps
also formation of polarons in the vicinity of charge moving through DNA.
[
36
,
37
,
39
]
Long-
range, shallowly distance-dependent DNA CT thus depends on several factors and cannot be
adequately explained by a single mechanism. Charge migration through DNA likely occurs
through a variety of pathways, with some delocalization over DNA domains gated by the
DNA conformational dynamics.
[
38
,
46
]
Though these studies have demonstrated a variety of mechanistic processes at work during
DNA CT, they also consistently demonstrate its key properties under all conditions assayed.
DNA charge transport occurs over long molecular distances
[
29
31
,
38
,
46
48
]
and exhibits a
shallow dependence on distance traveled.
[
29
31
,
40
,
46
49
]
Charge transfer through DNA
furthermore requires coupling of electron donors and acceptors into the
π
-stack and is
attenuated in the presence of perturbations in the base stacking interactions, such as base-
pair mismatches,
[
23
,
29
31
]
bulky lesions in the duplex sequence,
[
50
]
or severe structural
distortion of the helical stacking.
[
51
,
52
]
These fundamental characteristics all suggest that
Nature may harness DNA CT during the search for and subsequent repair of DNA damage
sites, which attenuate charge transport.
3. DNA-binding Proteins contain [4Fe4S] Clusters
Duplex DNA with well-stacked bases is thus a strikingly effective mediator of charge
transport, but what proteins serve as the electron donors and acceptors? In order to utilize
DNA CT for long-range redox reactions, electron donors and acceptors must both possess a
metal center capable of one-electron transfer reactions and bind duplex DNA such that their
redox centers are coupled into the
π
-stacked bases. Cunningham and coworkers initially
discovered
[
53
]
in 1989 that the base excision repair (BER) glycosylase Endonuclease III
(EndoIII) in
E. coli
, which repairs oxidized pyrimidine bases
[
54
]
in genomic DNA, contains
a [4Fe4S] cluster cofactor. Several other DNA repair proteins including BER glycosylase
MutY (
E. coli
),
[
55
]
family 4 Uracil-DNA glycosylase from thermophiles (UDG),
[
56
]
Nucleotide excision repair helicase XPD (
S. acidocaldarius
) (
Sa
XPD),
[
57
]
and R-loop
maturation helicase DinG (
E. coli
)
[
58
]
have since been discovered to contain [4Fe4S]
clusters. The transcription factor SoxR, which binds DNA and activates transcription of
soxS
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genes to defend the cell during oxidative stress, has additionally been shown to contain a
[2Fe2S] cluster cofactor.
[
59
]
Remarkably eukaryotic DNA polymerases and DNA primase
were also recently shown to contain[4Fe4S] centers.
[
60
,
61
]
Iron-sulfur cofactors, such as the rhomboid [2Fe2S] cluster and cubane [4Fe4S] cluster
(Figure 2), are often agents of redox chemistry in biology, with a tunable range of potentials
from 300mV to below −500mV vs. NHE.
[
62
]
These species likely arose during prebiotic
conditions on Earth, when an abundance of ferrous iron and sulfide would have been present
in the atmosphere.
[
63
]
These cofactors are generally coordinated by conserved cysteine
motifs, and require a large group of proteins to assemble and load the clusters into their
recipient proteins.
[
1
,
64
,
65
]
Incorporation of an iron-sulfur cofactor into a protein is a complex
and metabolically expensive task for cellular machinery, requiring biogenesis and assembly
proteins, chaperone proteins for transport, and in the case of eukaryotes, a targeting complex
comprising of three proteins (CIA1, CIA2, and MMS19)
[
64
66
]
to load the cofactor into its
recipient protein.
The general process for iron-sulfur biogenesis and loading is summarized in Figure 2. In
bacteria, the ISC assembly machinery is generally responsible for cofactor generation,
though the NIF system has been identified as part of the maturation pathway for the iron-
sulfur containing enzyme nitrogenase and the SUF assembly system is implicated in
biogenesis during oxidative stress.
[
1
]
In eukaryotes, the ISC assembly machinery and
mitochondrial ISC export system are necessary for biogenesis of the cofactor
[
67
]
and the
cytosolic iron-sulfur assembly (CIA) machinery is required for cluster maturation.
[
68
,
69
]
Inorganic sulfur is acquired from a cysteine desulfurase (IscS) in bacteria or exported from
the mitochondria by Atm1,
[
67
,
70
]
then bound by a cysteine desulfurase (NFS1) in
eukaryotes. Cysteine desulfurase activity converts cysteine to alanine, generating a
persulfide which is then transported directly or indirectly to scaffold proteins (IscU in
bacteria, ISU in eukaryotes) for cluster assembly. Ferrous iron is donated by a protein
source, and electron transfer occurs to reduce S
0
to S
2−
present in iron-sulfur clusters. After
the
de novo
biosynthesis of a labile cluster coordinated by scaffold protein cysteine residues,
the cluster-containing scaffold associates with chaperone proteins (HscA/HscB in bacteria,
HSC20/HSPA9 in eukaryotes). ATP hydrolysis likely drives a conformational change in
order to keep the labile cluster shielded during delivery to the recipient protein,
[
1
,
64
]
and
direct or indirect transfer of the cofactor to the recipient protein occurs.
Creation and incorporation of iron-sulfur clusters into proteins is thus a metabolically
expensive task requiring the engagement of several protein systems. Placing an iron-
containing cofactor in a DNA-binding protein could additionally place the bound nucleic
acid at risk of damage. A labile ferrous iron from the cofactor could react with hydrogen
peroxide in the cellular environment; this Fenton chemistry creates reactive oxygen species
(Figure 2) which could damage nearby DNA bases. Why then does Nature spend the
requisite energy incorporating a redox-active inorganic cofactor into a DNA-processing
enzyme?
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4. Platforms to Study DNA CT
The development of new methods was necessary to test whether DNA conducted charge in
solution at ambient temperatures. Certainly, assessing potential biological roles for DNA CT
chemistry requires experiments to be performed in aqueous solution using physiologically
relevant conditions. Key properties of DNA charge transport have now been characterized
extensively, through the development of several robust and versatile
platforms.
[
19
27
,
50
52
71
81
]
DNA CT has been studied in the excited state and the ground
state, as well as using single- molecule assays. Across all of the platforms developed,
consistent features of DNA CT are observed: (
i
) charge transport through duplex DNA is
dependent on the coupling of redox donors and acceptors to the
π
-stacked bases; (
ii
) DNA
CT can occur over long molecular distances with shallow distance dependence; and (
iii
)
DNA CT is attenuated in the presence of even minor perturbations in
π
-stacking interactions
between the bases.
Our laboratory initially assayed the charge transport properties of DNA using photophysical
studies, employing DNA-intercalating redox donors and acceptors. We covalently appended
metal complexes such as [Ru(phen)
2
dppz]
2+
(phen= 1,10 phenanthroline, dppz =
dipyrido[3,2-
a
:2
,3
-
c
]-phenazine) and [Rh(phi)
2
(phen)]
3+
(phi = 9,10-phenanthrenequinone
diamine) to a short, 15-bp oligonucleotide.
[
19
]
These metal complexes intercalate into the
duplex DNA bases and upon irradiation, [Ru(phen)
2
dppz]
2+
acts as a luminescence donor,
which is quenched through an intra-duplex mechanism by the [Rh(phi)
2
(phen)]
3+
acceptor.
(Figure 3) When an oligonucleotide modified with the [Ru(phen)
2
dppz]
2+
donor and
[Ru(phen)
2
(phen
)]
2+
(phen
= 5-amido-glutaric-acid-1,10-phenanthroline), a poor DNA
intercalator, is irradiated, luminescence is no longer quenched, suggesting a mechanism
dependent on intercalation, which is required for coupling of the donor and acceptor into the
DNA duplex.
To investigate further the mechanism of charge transport through DNA bases, we assayed
luminescence quenching of fluorescent base analogues 2-aminopurine (A
2
) and 1,
N
6
-
ethenoadenine (A
ε
) by guanine (G) or deazaguanine (Z)
[
25
]
which possess low enough redox
potentials to act as acceptors.
[
82
]
This assembly allowed for investigation of DNA CT
without potential constraining effects of covalent intercalating metal complex donors and
acceptors on the DNA helix structure. A long-range, shallowly distance-dependent
mechanism of quenching for A
2
, which couples well into the DNA
π
stack, was observed.
This mechanism was not observed, however, for the bulkier A
ε
analogue, which is less
capable of
π
-stacking and thus less effective in coupling into duplex DNA.
[
25
]
Fluorescence
quenching of a DNA-intercalated ethidium donor by a [Rh(phi)
2
(bpy)]
3+
acceptor through a
17-bp DNA duplex, moreover, is fivefold less efficient in the presence of a single CA base
pair mismatch, relative to a well-matched duplex.
[
23
]
These results together suggest that
well-stacked DNA bases are essential for charge transfer through DNA; perturbing the
π
-
stacking interactions severely attenuates DNA CT.
Using DNA-intercalating photooxidants, we have also observed DNA charge transport or
DNA damage generated at a distance. An intercalating oxidant such as [Rh(phi)
2
(bpy
)]
3+
(bpy
= 4-butyric acid, 4
-methylbipyridine) can be tethered to one end of a 63-bp duplex
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DNA substrate and photoexcited, oxidizing low-potential guanine bases at the 5
-end of a
5
-GG
-3
doublet sequence.
[
26
]
(Figure 3) Photolysis leads to the oxidation product 7,8-
dihydro-8-oxo-2
-deoxyguanosine (8-oxo-G) to form in a DNA-mediated manner, observed
up to 200 Å from the site of intercalation. These reactions also have been demonstrated to
occur on the picosecond timescale;
[
27
]
charge can move 10 times the single-step tunneling
distance through protein in a miniscule fraction of the time!
Additional platforms have been used to study DNA CT in the excited state, elucidating the
mechanisms at play during a DNA-mediated charge transport event. Injection of charge
through photoexcitation of a 4
-acylated nucleotide at the 3
-end of a duplex oligonucleotide
oxidizes low-potential guanine bases at a distance and corroborates the shallow distance
dependence observed in experiments measuring guanine damage.
[
40
]
Theoretical modeling,
in conjunction with photooxidation of DNA hairpins bridged with a stilbene-
derivative,
[
83
85
]
also demonstrate that DNA CT occurs at a distance and these results also
could not be explained exclusively by a coherent superexchange mechanism.
[
33
]
Photoexcited anthraquinone, (Figure 3), tethered to one end of a duplex DNA substrate, can
also be used to damage guanine bases in an oligonucleotide sequence.
[
36
,
38
,
39
,
46
]
Schuster
and coworkers employed this platform, along with computational modeling of DNA
interactions with Na
+
counterions and water molecules in solution, providing results
consistent with environment-dependent partial charge delocalization on DNA.
[
39
]
After observing rapid, long-distance, and mismatch-sensitive DNA CT in the excited state,
methods to study DNA CT in the ground state were also developed. Our laboratory has
utilized both a single-molecule
[
79
,
80
]
and a multiplexed electrode
[
76
78
]
platform to study
ground-state DNA CT. In the single-molecule platform, a gap is first chemically etched into
a single-walled carbon nanotube (SWCNT). A molecule of single-stranded DNA (ssDNA)
modified with an amine group at both ends, is covalently attached through amide coupling to
the oxidatively etched SWCNT, functionalized at the ends with a carboxylic acid group.
[
79
]
A well-matched complementary strand, or a strand containing a single-base mismatch, is
then annealed to the ssDNA bridging the etched nanotube. This assembly creates a circuit
(Figure 4) that allows for measuring the conductance of a single DNA molecule relative to
the nanotube. The source-drain current measured using this platform demonstrates that
charge flows through well-matched, but not mismatched, DNA duplexes. Site-specific
restriction enzyme
[
79
]
and methyltransferase activity
[
80
]
have moreover been demonstrated
on duplex DNA substrates in this setup; the DNA circuit is biologically accessible.
Additional single-molecule platforms to study ground-state DNA CT, based on readout from
electrochemical atomic force microscopy (AFM) or scanning tunneling microscopy
(STM),
[
47
,
48
,
71
]
have been designed and used to measure conductance through an
oligonucleotide. Tao and coworkers
[
71
]
measured passage of current at different applied
voltages through a single 8-bp dsDNA substrate. The DNA is modified at both ends with
alkanethiol groups, with one end covalently attached to an Au surface and the other end free
to link covalently to a gold STM tip brought into close contact with the DNA-modified gold
surface. This study demonstrated consistently high conductance of duplex DNA and a
shallow distance dependence for DNA CT, relative to electron transfer through
σ
-bonded
species.
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Electrochemical AFM studies of conductance through a single DNA molecule have also
been performed by Porath and coworkers,
[
47
,
48
]
using the platform design in Figure 4. A
single strand of DNA (26 nucleotide length) is attached to a gold surface through an
alkanethiol moiety. This strand is hybridized to a complementary strand, which is covalently
attached to a gold nanoparticle through an alkanethiol moiety. An electrochemical AFM tip
can then contact the gold nanoparticle on the end of the duplex DNA substrate and measure
conductance through the oligonucleotide. This platform demonstrated high conductance
through 26 base pairs of DNA,
[
47
]
consistent with other single molecule studies.
[
71
,
79
,
80
]
It
was additionally used to demonstrate that duplex DNA, but not single-stranded DNA, is able
to conduct charge.
[
48
]
The SWCNT circuit, STM, and electrochemical AFM platforms for studying ground-state
DNA CT through a single molecule are complemented by studies using the multiplexed
DNA-modified gold electrode platform.
[
76
78
]
(Figure 4) This high-throughput system
facilitates obtaining reproducible electrochemical readouts for ground-state, aqueous DNA
CT under different conditions (well-matched versus mismatched duplex DNA, for example),
all on a single electrode surface. DNA-modified gold surfaces are first constructed by
incubating a duplex DNA substrate with an alkanethiol moiety at one end on the Au(1,1,1)
surface, allowing a covalent thiol-gold attachment to form. The DNA molecules then form a
densely packed self-assembled monolayer on gold, oriented at a 45° angle relative to the
electrode surface, as shown using AFM.
[
86
]
After the gold surfaces are incubated with
alkanethiol-modified DNA and any remaining bare gold is passivated using
β
-
mercaptohexanol,
[
76
78
]
the DNA-modified surfaces become the working electrode in a
three-electrode cell setup, with a silver/silver chloride reference and a platinum auxiliary
electrode. Electrochemical techniques such as cyclic voltammetry (CV), square wave
voltammetry scanning, and bulk electrolysis can be used to monitor the DNA-mediated
passing of charge between the gold surface and a redox-active species at the distal end of the
DNA duplex, over a physiologically relevant range of potentials.
[
24
,
50
,
51
,
72
78
,
82
,
86
94
]
The multiplexed electrode system (Figure 4) was adapted from a single-surface DNA-
modified electrode platform developed in our laboratory
[
24
,
87
]
and allows for scanning of 16
DNA-modified electrodes on a single surface. The gold electrodes, each with an area of
2mm
2
, are arranged in four quadrants on the silicon chip.
[
76
]
These quadrants are divided
physically from one another in the multiplex chip setup for electrochemistry, so that as many
as four different DNA substrates can be incubated on one chip. In this manner, facile,
reproducible readouts for a new experimental condition can be obtained alongside control
experiments; there is no longer a need to measure two conditions for the same experiment on
different gold surfaces.
Using our DNA-modified electrode platform, we have observed properties of DNA CT
consistent with the results of photophysical and single-molecule studies, obtaining readouts
from both redox-active dyes such as Nile Blue
[
51
,
77
,
94
]
and DNA-bound enzymes with
redox-active [4Fe4S] clusters (
vide infra)
.
[
72
75
,
78
,
88
,
89
,
92
]
DNA-mediated redox signals on
the gold electrode are contingent upon coupling of the redox-active species to the
π
-stacked
bases.
[
23
,
91
]
DNA CT additionally has been observed over long distances on this platform; a
DNA-mediated signal for the redox probe Nile Blue was observed over 100 bp (34nm) of
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DNA. The signal through 100 bp of duplex DNA remarkably displays the same kinetics and
mismatch sensitivity as a redox signal through 17 bp of DNA. The distance dependence of
DNA CT in the ground state is shallow; the rate-determining step is electron tunneling
through the alkanethiol linker.
[
77
]
Finally, DNA CT has been shown to be sensitive to
perturbations in the base-stacking interactions using this platform; DNA-mediated
electrochemical signals are attenuated in the presence of base pair mismatches,
[
23
,
76
,
77
,
95
]
bulky lesions,
[
50
]
and structural distortions such as bending of the upright helix through
binding of TATA-binding protein.
[
51
,
52
]
The duplex DNA substrates on the multiplex electrode surfaces, importantly, are present in a
biologically accessible conformation. We have demonstrated, for example, that when a
binding site for restriction enzyme
RsaI
is engineered into the 100-bp DNA substrate on the
multiplex chip platform,
RsaI
will bind the DNA and cut at the restriction site when
incubated on the Au electrode.
[
77
]
We have additionally demonstrated methyltransferase
activity on these electrodes, in a setup with a signal-on detection based on the readout from a
Nile Blue signal.
[
94
]
These enzymatic activity readouts were based on redox probe signals,
but we have also been able to monitor the DNA-bound electron transfer activity of enzymes
with [4Fe4S] clusters using these electrodes. Incubation of [4Fe4S] repair helicases
Sa
XPD
[
92
]
and DinG
[
73
]
(
E. coli
) on DNA-modified gold electrodes produces a large, ATP-
dependent DNA-mediated redox signal, associated with the coupling of the [4Fe4S] cofactor
to the duplex DNA; interestingly, coupling increases with ATP-dependent helicase activity.
Many research groups have thus developed systems which have allowed for characterization
of the electronic properties of DNA along with effective methods to test how DNA charge
transport may be used in biology. This long-range, rapid chemistry is sensitive to
perturbations in the base-stacking interactions, and these characteristics have consistently
and reproducibly been seen in the ground state and the excited state, on single-molecule,
ensemble, and multiplexed platforms. Building on the foundation of these robust
in vitro
methods, we can therefore investigate the DNA-mediated charge transfer properties of
biological systems and how this chemistry is utilized within the cell.
5. DNA Charge Transport in Biology: Initial Observations
After characterizing the fundamental properties of DNA charge transport on a molecular
level using chemical and physical methods, we next focused on answering the question: can
DNA CT operate in a cellular environment, modulating reactions at a distance as it does
in
vitro
? The first important question to answer was whether DNA CT occurred in cell
nuclei,
[
96
]
where genomic DNA is produced and stored. Upon treatment of HeLa cell nuclei
with the DNA-intercalating photooxidant [Rh(phi)
2
DMB]
3+
(DMB = 4,4
-dimethyl-2,2
-
bipyridine), we observed long-range oxidation of guanine bases at the 5
-positions in 5
-
GG-3
doublet and 5
-GGG-3
triplet sequences upon irradiation of the samples to
photoexcite the Rh(III) compound. Damage at 5
-guanines in 5
-GGG-3
and 5
-GG-3
sites, a signature of DNA-mediated one electron oxidation, was furthermore demonstrated in
mitochondria,
[
97
]
upon irradiation of a Rh(III) intercalating photooxidant. Long-range
oxidation of low-potential guanine sites occurred in cells, affecting regions to which proteins
were known to bind and those free of proteins;
[
96
]
for example the binding of a transcription
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factor
[
96
]
did not inhibit DNA-mediated damage of guanine residues; charge can migrate
through bases as long as the helical structure and stacking interactions are not distorted.
We also investigated whether DNA CT could occur through duplex DNA bound in a
nucleosome core particle (NCP), the fundamental unit of DNA packing in eukaryotic cells.
(Figure 5) An octameric complex of histone proteins binds and wraps ~150 bp of duplex
DNA in an NCP structure, which then is further assembled into higher-order structures in the
nucleus.
[
98
,
99
]
Since a large portion of the eukaryotic genome is bound in these structures,
which curves the helix gently around the histone core, it is relevant to ask whether CT can
occur through histone-bound DNA to affect genome-wide reactions. Using the DNA-
intercalating [Rh(phi)
2
(bpy)]
3+
photooxidant tethered to DNA in the presence and absence
of histones, we observed long-range oxidative damage to 5
-guanine bases in 5
-GG-3
doublets and 5
-GGG-3
triplets.
[
100
]
As DNA CT occurs in histone-bound, packaged DNA,
it is a feasible mechanism for genome-wide signaling and sensing of oxidative stress
conditions in eukaryotic cells. This result also confirms further that DNA CT can occur
through protein-bound DNA as observed in HeLa cell nuclei, as long as the
π
-stacking base
interactions are maintained.
Protein binding which does perturb the coupling of the
π
-stacked bases, however, can
modulate charge transfer through DNA. TATA-binding protein (TBP), for example, is a
ubiquitous transcription factor that bends DNA approximately 80° when it binds.
[
101
]
We
have demonstrated using our DNA electrochemistry platform that TBP attenuates DNA CT
in binding and severely kinking the double helix, disrupting base pair
π
-stacking
interactions.
[
51
]
TBP binding and kinking of the DNA double helix therefore allows for
modulation of redox reactions at a distance by changing the CT properties of protein-bound
DNA. This modulation is reversible, occurring only when TBP is bound to a segment of
duplex DNA. It is dependent on the structural properties of the DNA helix in the presence or
absence of a binding event and suggests that proteins, in modulating DNA CT, can play a
range of regulatory roles during the cell cycle.
One important dynamic condition in the cellular environment which requires a biological
response is oxidative stress.
[
102
]
SoxR is a transcription factor in enteric bacteria containing
a [2Fe2S] cluster and is responsible for binding the
soxS
promoter in the bacterial genome to
activate transcription of genes to respond to oxidative stress in the cell.
[
102
,
103
]
SoxR binds
DNA as a dimer, and the apo-form, which does not contain the cluster, binds with a similar
affinity as the holoenzyme containing the [2Fe2S] cofactor.
[
104
]
The transcriptional
activation activity, however, is dependent on the cluster.
[
105
]
We showed using DNA
electrochemistry that SoxR undergoes a potential shift of +490mV when bound to DNA,
suggesting that the DNA-bound protein is generally in the reduced [2Fe2S]
+
state but can be
oxidized to the [2Fe2S]
2+
state at cellular potentials where oxidative stress arises.
[
52
]
SoxR
is capable of DNA-mediated redox activity when bound to DNA, but can this transcription
factor respond to oxidative stress at a distance in a DNA-mediated manner?
To investigate whether DNA charge transport can modulate the redox state, and therefore the
transcriptional activity, of SoxR at a distance, we employed DNA-intercalating
photooxidants. We demonstrated with noncovalently bound [Ru(phen)(dppz)(bpy
) ]
2+
O’Brien et al.
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Isr J Chem
. Author manuscript; available in PMC 2017 January 11.
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