DNA Charge Transport within the Cell
Michael A. Grodick
,
Natalie B. Muren
, and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena
CA 91125
Abstract
The unique characteristics of DNA charge transport (CT) have prompted an examination of roles
for this chemistry within a biological context. Not only can DNA CT facilitate long range
oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and
participate in long range redox reactions using DNA CT. DNA transcription factors with redox-
active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT
chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA
CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize
this chemistry include an interesting and ever-growing class of DNA-processing enzymes
involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S
clusters. DNA repair enzymes containing 4Fe-4S clusters, that include Endonuclease III (EndoIII),
MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active
when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at
long range via DNA CT. Interactions between DNA and these proteins in solution, in addition to
genetics experiments within
E. coli
, suggest that DNA-mediated CT can be used as a means of
cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in
finding DNA damage, even within cells. Based on these data, we can consider also how DNA-
mediated CT may be used as a means of signaling to coordinate DNA processing across the
genome.
Our laboratory has focused on studies of DNA-mediated charge transport (CT). This
chemistry offers a means to carry out redox chemistry at a distance and provides a sensitive
reporter on the integrity of the intervening DNA. In photophysical, biochemical, and
electrochemical experiments we have learned general features of this chemistry. Perhaps
uniquely, DNA CT can occur by transport through the base pair stack over long molecular
distances. But importantly, this long distance redox chemistry can only occur if the DNA
helix is well stacked; anything that interrupts that stacking turns off CT. Thus the chemistry
offers a means to effect long range redox signaling as long as the integrity of the DNA
duplex is intact. The uniqueness of this chemistry thus begs the question: Is DNA CT
utilized within the cell?
Here we describe experiments to begin to probe how this chemistry may be utilized within
the cell. DNA CT chemistry is important to consider in the context of how DNA may be
*
to whom correspondence should be addressed at jkbarton@caltech.edu.
HHS Public Access
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Biochemistry
. 2015 February 3; 54(4): 962–973. doi:10.1021/bi501520w.
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damaged under conditions of oxidative stress and how that damage is sensed and repaired.
Increasingly, proteins involved in DNA processing have been found to contain 4Fe-4S
clusters, cofactors generally thought to carry out redox reactions within the cell. Moreover
DNA CT can promote redox chemistry over long molecular distances, potentially providing
a means for signaling across the genome. Furthermore, because DNA CT reports on the
integrity of the DNA duplex, this signaling can reflect whether the intervening DNA is
damaged and in need of repair, whether DNA processing needs to begin, to be stalled or to
be increased.
We begin by describing different experiments we have used to elucidate DNA CT
chemistry. What are the characteristic features of DNA CT? Within that framework, we next
describe experiments carried out on DNA-binding proteins to explore how this chemistry
may be utilized. The work presented is not meant to be an exhaustive survey of the
literature.
1
–
4
Instead, the experiments described are intended to illustrate DNA CT
chemistry and particularly a role for proteins containing 4Fe-4S clusters in carrying out
signaling using this chemistry. These studies thus present a framework for considering how
DNA CT may be used for genomic signaling.
Platforms to Study the Characteristic Features of DNA CT
The conductivity of DNA arises most fundamentally from its continuous,
π
-stacked core of
aromatic bases that extends down the helical axis. In fact, this conductivity was first
predicted from early DNA structural studies which revealed striking similarities to sheets of
graphite, both in
π
-stacking and interplanar spacing.
5
However, unlike graphite and other
π
-
stacked solids, DNA is a dynamic, macromolecular array that functions in solution. This
critical difference gives rise to several defining characteristics of DNA CT, and thus studies
to probe this chemistry must be performed using platforms with aqueous conditions in which
the dynamic motions of the DNA bases are unrestricted. In our laboratory we have
established three such general platforms to study DNA CT in DNA duplexes from different
vantage points: free in solution, tethered to electrode surfaces, and as single molecules.
2
Across these platforms we have observed several essential, conserved features of this
chemistry, including (i) electronic coupling of the donor and acceptor to the DNA
π
-stack is
required; (ii) even slight disruptions to the DNA
π
-stack between the donor and acceptor
inhibit DNA CT; and (iii) the distance dependence of DNA CT is very shallow. Here we
briefly summarize the platforms and fundamental studies that revealed these characteristics
and that are critical for DNA CT in biological systems.
Given the need for aqueous conditions to make biologically relevant measurements of DNA
CT, the first experiments to probe this chemistry were performed with free DNA duplexes in
solution containing pendant redox donors and acceptors. In one such construct, the
metallointercalators 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 diimine) were
covalently attached to either ends of a 15-mer DNA duplex as donor and acceptor handles,
respectively (Figure 1).
6
Photoexcitation of the donor results in luminescence that is rapidly
quenched by the acceptor via DNA CT. Importantly, coupling of the donor and acceptor to
the DNA
π
-stack is essential for this chemistry to proceed; substitution of Ru(phen)
2
(phen
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′
)
2+
(phen
′
= 5-amido-glutaric-acid-1,10-phenanthroline), a poor DNA intercalator, inhibits
quenching.
Similar results were obtained with a more native construct in which fluorescence quenching
of adenine base analogs by guanine allows for direct, base-to-base measurements of DNA
CT.
7
In these experiments, for which the base analogs 2-aminopurine (A
2
) or 1,N
6
-
ethenoadenine (A
ε
) were incorporated into 12-mer DNA duplexes, quenching of A
2
occurs
far more rapidly and over longer distances than quenching of A
ε
. This result is consistent
with the different structures of these analogs that allow for well integrated stacking of A
2
into the DNA helix and poor stacking of A
ε
. Beyond the importance of electronic coupling
to the
π
-stack to participate in DNA CT, solution-based platforms also provided the first
indication of the exquisite sensitivity of this chemistry to perturbations along the
π
-stacked
path; the introduction of even a single base mismatch between the donor and acceptor
severely decreases the quenching yield.
8
Studies of base-base CT also taught us important
lessons about the timescales and dynamics of DNA CT. DNA CT occurs on the picosecond
timescale but is gated by the motions of the bases.
9
In order to relate these observations more directly to DNA CT in biological systems, it was
necessary to design platforms that allow ground state measurements of DNA CT, while still
maintaining the DNA in an aqueous, buffered environment. In one such platform, a single
DNA duplex is made to covalently span an etched gap in a carbon nanotube circuit.
10
Current flow through this device reports directly on DNA CT efficiency (Figure 2). By
cycling the type of DNA incorporated into the device from well matched DNA to DNA with
a single base mismatch to well matched DNA again, current flow is turned on, then off, then
on again, respectively. This ground state, single molecule platform provides more direct
measurements of DNA CT and confirms the high sensitivity of this chemistry to even minor
structural disruptions of the
π
-stack.
In a second ground state platform, DNA functionalized with an alkanethiol linker is allowed
to self-assemble vertically as a film on a gold electrode surface (Figure 3). These DNA-
modified electrodes can then be used to monitor DNA CT electrochemically between the
electrode and a redox-active probe molecule bound at the distal end of the DNA.
11
,
12
Taking
advantage of the inherent capacity of electrochemistry for multiplexing, we developed 16-
electrode chips to facilitate side-by-side analysis of multiple samples and controls, thereby
opening the door for even more complex investigations of DNA CT (Figure 3). Importantly,
the same characteristics of DNA CT that are observed with free DNA in solution are
observed with DNA-modified electrodes; electronic coupling of the redox probe to the DNA
π
-stack is essential to observe a DNA-mediated redox signal
13
and DNA CT is disrupted by
a variety of biologically significant perturbations to the DNA
π
-stack, including intervening
base mismatches,
13
–
16
base lesions,
17
and structural distortions caused by protein binding
and activity.
18
,
19
Critical for studying DNA CT in biological systems, DNA-modified electrodes also make it
possible to measure the DNA-bound potentials of proteins with redox-active cofactors such
as iron-sulfur clusters (Figure 3).
20
–
25
Like synthetic redox probes, DNA CT to DNA-bound
proteins show the same coupling requirement to the
π
-stack
22
,
23
and the same sensitivity to
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intervening structural disturbances, such as base mismatches and lesions.
20
,
24
,
25
Thus DNA-
modified electrodes allow for the identification and characterization of proteins that have the
capacity to participate in DNA CT chemistry in living organisms.
In addition to illustrating these first two characteristics of DNA CT chemistry, these diverse
platforms were also used to probe a third critical parameter for understanding the role of
DNA CT in biology:
how far can DNA effectively conduct charge?
This distance
dependence was first studied with free DNA in solution, using a covalently attached
metallointercalator photoxidant that, upon irradiation, induces DNA-mediated, long-range
guanine oxidation.
26
,
27
By biochemical sequencing, this damage is observed at the 5
′
-G of
guanine doublets, the site of lowest oxidation potential. Importantly, and consistent with a
DNA CT mechanism, the degree of coupling of the photoxidant to the
π
-stack and the
integrity of the intervening
π
-stack were found to be far greater determinants of oxidation
yield than the distance of the oxidation site from the photoxidant. In fact, over the longest
distance measured, 20 nm or 60 bp, the oxidation yield was unaffected by the separation
distance.
27
Using multiplexed, DNA-modified electrodes, ground state DNA CT to a distal, covalent
redox probe was measured over an even longer distance of 34 nm or 100 bp (Figure 3).
16
For these experiments, the multiplexed chip platform was crucial for enabling the side-by-
side comparison of DNA CT in short and long DNA duplexes. Remarkably, DNA CT over
34 nm yields the same redox signal size and the same signal attenuation from the
incorporation of a single base mismatch as DNA CT in much shorter, 6 nm or 17 bp
duplexes. Over both 34 nm and 6 nm, the rate of DNA CT is limited by the electron
tunneling rate through the alkanethiol linker that attaches the DNA to the gold surface.
16
,
28
Thus, the distance dependence of DNA CT is very shallow, allowing efficient charge
conduction by DNA over distances that challenge the physical limits of these
in vitro
platforms for measuring this chemistry. Indeed, we have not yet determined an upper limit
in distance for DNA CT, only that over these 100 base pair distances, CT is efficient and no
decay in yield is observed. After establishing these conserved, structurally derived
characteristics of DNA CT, (i) required electronic coupling to the
π
-stack, (ii) high
sensitivity to intervening structural perturbations of the
π
-stack, and (iii) a capacity to
transport charge over very long distances, the clear next step was to utilize this foundation to
consider a role for this chemistry in living cells.
General Observations of DNA CT in Biological Systems
Initial experiments probed broadly the possibility that DNA CT might play a role in
biological systems and sought to identify the cellular players that have the capacity to
engage in this chemistry.
Can cellular DNA participate in DNA CT? Can DNA-binding
proteins participate in DNA CT?
From this beginning point, the ways in which DNA CT
may be exploited by these players were then probed including the funneling of charge
through cellular DNA to concentrate damage at distant sites throughout the genome and the
sending and receiving of DNA-mediated charge by DNA-binding proteins as a means to
sense and respond to oxidative stress. These general observations set the stage for our
current, extensive studies on whether DNA-bound proteins may use DNA CT to signal to
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each other across the genome in order to achieve more efficiently a variety of logistically
challenging biological tasks.
The measurement of long-range oxidative damage to DNA in vitro
26
,
27
set the stage to
investigate this phenomenon in cellular DNA (Figure 4). Reactive oxygen species pose a
constant threat to the integrity of the genome, making it critical for cells to reduce the net
impact of inevitable damage. One strategy is to promote the accumulation of holes at
specific regions of low oxidation potential, namely tracts of multiple guanine bases, in order
to concentrate damage and spare the majority of the DNA.
29
The possibility that DNA can
facilitate such long-range funneling of damage via DNA CT was investigated in several
native cellular environments including isolated HeLa cell nuclei,
30
nucleosome core
particles,
31
and mitochondria.
32
,
33
In these studies, oxidative damage was induced by
photoexcited [Rh(phi)
2
(bpy)]
3+
(bpy = 2,2
′
-bipyridine), a DNA intercalator and potent
photooxidant (Figure 4). A comparison of the binding sites of the metal complex with the
locations of guanine damage showed significant separation, necessitating some form of
controlled charge migration. Additionally, damage was observed specifically at the 5
′
-
guanine of guanine repeat sites, a hallmark of DNA CT. Thus, these studies verified that
long-range DNA CT can indeed take place in a complex and congested native organelle
environment and can divert DNA damage to distant reservoirs in the genome.
34
Given the confirmation that cellular DNA can facilitate DNA CT across long distances, the
next step was to determine whether DNA-binding proteins can access, and potentially
exploit, this chemistry. A logical starting point was to consider redox-active proteins that
protect the cell against oxidative stress and thus must be able to sense and respond to
oxidative threats. The first such protein investigated was SoxR, a bacterial transcription
factor that, when activated, induces the transcription of a battery of genes involved in the
oxidative stress response (Figure 4).
35
SoxR generally remains bound to DNA as a dimer in
the cell and each monomer contains a [2Fe-2S] cluster that critically allows it to sense and
respond to oxidative stress; oxidation of the cluster results in an up to 100-fold increase in
transcription of its stress response genes.
36
But how is the cluster oxidized?
Electrochemistry of SoxR bound to DNA-modified electrodes revealed that redox activity of
the [2Fe-2S] clusters can be accessed by DNA CT. Additionally, the potential of the cluster
shifts by positive ~500 mV upon DNA-binding, thereby modulating the capacity of SoxR to
function as an oxidative stress sensor.
37
To investigate whether SoxR can be activated by
oxidative DNA damage from a distance, the DNA intercalating photooxidant
[Rh(phi)
2
(bpy)]
3+
was covalently attached to a DNA duplex, 80 bp from the SoxR binding
site (Figure 4). Upon photoexcitation of the metal complex, which injects electron holes into
the DNA and generates distant guanine radicals, transcription of SoxR-regulated genes is
activated.
38
This result indicates that SoxR can utilize DNA CT to sense oxidative damage
efficiently across long molecular distances in the genome and activate the appropriate
protective response.
Recent studies were conducted on bacterial Dps and human p53, other DNA-binding redox-
active proteins that are also involved in responding to oxidative stress. Dps, a bacterial mini-
ferritin which uses ferroxidase activity to protect DNA in pathogenic bacteria from reactive
oxygen species, was studied to determine if it can wield this capability from a distance, via
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DNA CT (Figure 4). DNA-bound, ferrous iron-loaded Dps, but not apo-Dps or ferric iron-
loaded Dps, was observed to neutralize distant guanine radicals formed by a distally bound,
covalent photoxidant, confirming its potential to use DNA CT for cellular protection.
39
Similarly, human p53, a transcription factor that decides the fate of human cells under
stressful cellular conditions, was found to respond to distal hole injection into DNA by a
covalent anthraquinone photoxidant.
40
,
41
Specifically, oxidation of multiple cysteine
residues to disulfide bonds within p53 causes dissociation from specific DNA promoter
sequences. Importantly, p53 binding sites with lower oxidation potential (ie. sites with
guanine doublets and triplets) show more dissociation of p53 in response to the oxidative
insult.
41
Thus, the redox sensitivities of p53-binding sites combined with long-range DNA
CT provide an efficient mechanism for p53 to regulate the expression of specific genes in
response to a genome-wide report of oxidative stress.
Collectively, these examples demonstrate that diverse DNA-binding proteins, with a variety
of redox-active cofactors, have the ability to take advantage of DNA CT chemistry in order
to achieve more efficiently a variety of challenging cellular tasks. This work has fueled more
in-depth studies into perhaps the most intriguing and powerful question that arises from
these initial observations of DNA CT in biological systems:
Do DNA-bound proteins use
DNA CT to signal to each other?
Signaling among DNA repair glycosylases containing 4Fe-4S clusters
A striking number of DNA-processing enzymes in both prokaryotes and eukaryotes have
been found to contain 4Fe-4S clusters. This iron co-factor was first associated with DNA
repair enzymes when a 4Fe-4S cluster was discovered in
E. coli
Endonuclease III (EndoIII),
a DNA glycosylase from the base excision repair (BER) pathway.
42
–
44
Other BER enzymes,
such as
E. coli
MutY and homologous BER enzymes from other organisms, were also
shown to contain 4Fe-4S clusters.
45
,
46
The 4Fe-4S cluster in these enzymes resides near the
DNA binding interface and the clusters do not play a redox role in catalysis. Originally, the
role of these iron-sulfur clusters was thought to be solely structural. While iron-sulfur
clusters often serve a redox role in other classes of enzymes, a redox role for the clusters in
the DNA glycosylases was first rejected, since for EndoIII the reduction potential of the
[4Fe-4S]
2+/1+
couple was found to be < −400 mV vs. NHE based on measurements using
redox mediators.
43
,
44
Additionally, while the cluster in MutY is required for DNA binding,
the overall structure of MutY is not affected by the absence of the cluster.
46
Our laboratory hypothesized instead that the 4Fe-4S clusters in these proteins are used for
DNA-mediated redox signaling.
20
,
22
,
23
,
47
Importantly, measurements of redox
characteristics of the cluster were required with the protein bound to the DNA polyanion,
where the repair protein carries out its function. DNA-modified electrodes were thus used to
probe the redox characteristics of the 4Fe-4S cluster in DNA glycosylases. The first
important observations made were that the 4Fe-4S cofactors in DNA glycosylases can be
reduced or oxidized via long range DNA CT and binding to DNA shifts the reduction
potential of the 4Fe-4S cluster.
20
,
21
Cyclic voltammetry on DNA-modified gold electrodes
shows a reversible redox signal at a midpoint potential of ~80 mV vs. NHE (Figure 5). This
electrochemical signal is dependent on the DNA bases being well stacked. By introducing a
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single abasic site in the DNA substrate, the electrochemical signal is significantly
attenuated, consistent with the redox pathway from the electrode to the cluster being through
the DNA
π
-stack. Thus, the 4Fe-4S cluster of the protein appears to be well-coupled
electronically into the DNA base stack.
An array of EndoIII mutants have been studied electrochemically on DNA-modified
electrodes.
22
,
23
,
48
Interestingly, all of the mutants examined so far have the same DNA-
bound redox potential, though for many of the mutant enzymes, an increased or decreased
signal intensity as measured by cyclic voltammetry is observed, indicating the electronic
coupling of the mutant enzymes to the DNA base stack has changed (Figure 5) Of particular
interest is the CT-deficient EndoIII Y82A mutant, which has a significantly lower
electrochemical signal on DNA-modified electrodes compared to wild-type (WT) EndoIII,
though it maintains enzymatic activity comparable to that of the WT enzyme.
22
,
48
Direct electrochemical measurements of EndoIII on highly oriented pyrolytic graphite
(HOPG) with and without DNA revealed that upon binding to DNA, the redox potential of
the [4Fe-4S]
3+/2+
couple shifts negatively by ~200 mV.
21
The protein is thus activated
towards oxidation upon binding to DNA. The earlier studies suggesting Endo III to be
redox-inactive under physiological conditions still hold true, but not if the protein is bound
to DNA. This shift in potential moreover corresponds to a three orders of magnitude higher
affinity for DNA when the 4Fe-4S cluster is in the 3+ oxidation state relative to the 2+
oxidation state.
21
The increase in the binding affinity of the protein for DNA in the oxidized
form versus the reduced form is understandable given that higher oxidation states should be
preferentially stabilized when the 4Fe-4S cluster is in the vicinity of the polyanionic DNA
backbone.
The role of MutY and EndoIII is to prevent oxidized bases from causing mutagenesis, by
MutY excising bases that are mispaired with oxidized bases, or EndoIII directly excising
oxidized bases, prior to replication. A question that has yet to be definitively answered is
how these enzymes, thought to be at low copy number within cells, can locate and fix such
subtle damage scattered across the 4.6 megabase genome of
E. coli
before the organism
divides. The copy number of MutY is estimated to be ~30.
49
We have proposed that DNA
CT may be used by MutY, EndoIII, and other DNA-processing enzymes containing 4Fe-4S
clusters as a means to both scan the genome for damage and locate the damage as a first step
in DNA repair.
22
,
23
,
47
,
50
Our model for how DNA repair proteins with 4Fe-4S clusters use DNA-mediated CT as a
means of signaling to locate and repair damage, illustrated in Figure 6, is based upon many
of the redox characteristics discussed above.
22
When a DNA glycosylase is freely diffusing
in solution, its 4Fe-4S cluster is expected to be in the 2+ oxidation state. Upon binding to
DNA, the redox potential of the protein shifts and the 4Fe-4S cluster may be oxidized by
reactive oxygen species, other endogenous chemicals, or guanine radicals from a
distance.
51
,
52
If a second DNA glycosylase binds at a sufficient distance relative to the first
glycosylase to participate in a DNA-mediated redox reaction and the DNA between the two
proteins is undamaged, then the second glycosylase could transfer an electron through the
DNA base stack to reduce the distally bound protein. Once in the reduced form, this protein
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would have a lower binding affinity, dissociate from DNA, and take advantage of 3-D
diffusion to search the genome elsewhere. Importantly, this electron transfer event between
the two glycosylases would represent an effective scanning of the DNA integrity between
the two proteins. This scanning process would continue until the proteins bind near sites of
damage. Since DNA CT through the damaged DNA would be attenuated, any oxidized
protein would stay bound in the vicinity of the lesion, diffuse one-dimensionally to the
lesion, and process the lesion according to previously described mechanisms.
53
–
56
DNA CT
thus offers a mechanism for DNA glycosylases that contain 4Fe-4S clusters to use DNA-
mediated signaling
as a first step
in the search for damage, using DNA CT to scan the
genome and concentrate the repair proteins in the vicinity of damage.
An experiment using atomic force microscopy (AFM) was used to visualize protein-DNA
complexes in order to test directly if EndoIII redistributes onto DNA that contains
damage.
22
,
23
In this experiment, EndoIII was incubated with mixtures of DNA, one DNA
containing a single C:A mismatch, which attenuates DNA CT but is not a substrate for
EndoIII, the other DNA being fully matched and undamaged (Figure 7). The well-matched
DNA and mismatched DNA can be distinguished in the AFM given their different lengths.
The short strands (~1.9 kbps) are well-matched, while the long strands (~3.8 kbps) contain a
single C:A mismatch near the center of the strand. After incubation, what is found is that the
proteins do in fact redistribute to the long mismatched strands of DNA. Even though there is
only a single base mismatch in 3800 base pairs, the binding density of the protein is found to
be higher on the mismatched strand; as a control, where there is no mismatch in the long
strands of DNA, the protein density is the same on both the well-matched and mismatched
strands of DNA. Interestingly, we also tested the role of DNA CT in this experiment. As
discussed previously, EndoIII Y82A is a mutant of EndoIII that is CT-deficient as assessed
electrochemically, but has enzymatic activity comparable to that of WT EndoIII
22
,
48
; when
EndoIII Y82A is incubated with the mixtures of mismatched and well-matched DNA, there
is no observed redistribution. Adding hydrogen peroxide to the protein-DNA mixtures
during incubation increases the extent of redistribution suggesting that oxidative stress may
further drive redistribution to sites of damage.
23
Indeed, in subsequent experiments using
various mutants of EndoIII, we found a direct correlation between the proficiency in
carrying out DNA CT and the ability to redistribute onto the damaged strand. These results
strongly indicate that DNA-mediated redox signaling can be used by DNA glycosylases to
drive redistribution to the vicinity of DNA damage. But does this cooperative signaling
occur within a cell?
As predicted by the model, which is supported by the AFM results, if EndoIII and MutY use
DNA-mediated redox signaling as a first step to locate damage, then the activity within cells
of one of the glycosylases should be affected by the presence or absence of the other.
Genetics experiments were used to show that this indeed appears to be the case. A
lac
+
forward reversion assay using the CC104 strain of bacteria was used to measure the activity
of MutY upon silencing the gene encoding EndoIII,
nth
.
22
,
57
,
58
Within the CC104 strain of
bacteria, MutY prevents GC:TA transversions within the
lacZ
gene by removing adenines
placed opposite the oxidized lesion, 8-oxoG. Cells in which the GC:TA transversion has
occurred, termed
lac
+
revertants, can grow on media that contains lactose as the sole carbon
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source, providing a readout of MutY activity. When the
nth
gene was inactivated within
CC104, the number of
lac
+
revertants increased 1.5 to 2-fold reflecting a decrease in MutY
activity.
22
This result is consistent with signaling between EndoIII and MutY. Moreover,
mutants of EndoIII were expressed off of complementation plasmids in CC104 Δ
nth
to
provide evidence that this signaling occurs via DNA-mediated CT. When the CC104 Δ
nth
cells are complemented with EndoIII Y82A, the CT-deficient mutant, there is no rescue of
MutY activity. However, when the cells are complemented with EndoIII D138A, which is
catalytically inactive but CT-proficient, MutY activity is restored. These results in
combination with the results from AFM suggest that DNA glycosylases utilize DNA-
mediated redox signaling as a first step in locating DNA damage efficiently within cells.
Signaling enzymatic activity of DNA Helicases with 4Fe-4S Clusters
The evidence that DNA glycosylases may use 4Fe-4S clusters to participate in DNA CT in
order to localize to sites of damage in cells is intriguing, but importantly, other DNA-
processing enzymes have also been shown to contain 4Fe-4S clusters. For example, XPD is
an ATP-dependent helicase from the nucleotide excision repair pathway that is also part of
transcription-coupled repair.
59
,
60
In eukaryotes XPD is part of the TFIIH complex, which is
vital for both nucleotide excision repair and transcription. Mutations in XPD in humans can
lead to Xeroderma Pigmentosum and Cockayne’s syndrome. Recently, XPD along with an
entire family of helicases were predicted to contain 4Fe-4S clusters.
60
,
61
In 2008, three
separate crystal structures were published for XPD from three different species, confirming
the presence of the 4Fe-4S cluster in XPD. One structure was for XPD from
S.
acidocaldirus
59
, one from
S. tokodaii
62
, and one from
T. acidophilum
63
. Two of the crystal
structures contained the intact 4Fe-4S cluster when crystallized, while the third did not. Of
particular note is the observation that the global structure of XPD from
S. tokadaii
was not
perturbed even though it did not contain the 4Fe-4S cluster.
The XPD protein from
S. acidocaldarius
(SaXPD) was electrochemically characterized on
DNA-modified electrodes. Here, unlike in previous studies where a DNA duplex was
utilized, the substrate on the self-assembled DNA monolayers was a 20-mer double-stranded
duplex with a 9-mer 5
′
to 3
′
single-stranded overhang, which is a substrate that can be
unwound by helicases. Strikingly, the DNA-bound midpoint redox potential of SaXPD was
found to be ~80 mV vs. NHE, the same DNA-bound potential measured for the glycosylases
MutY and EndoIII from
E. coli
.
20
,
25
Moreover, since the substrate on the surface of the
electrode is a substrate that SaXPD can unwind, the effect of adding ATP to the solution was
studied. Upon adding ATP, the current intensity rises by 10–20%, and at a rate comparable
to the rate of ATP hydrolysis for SaXPD (Figure 8). Adding ATP
γ
S led to no increase in the
signal, indicating the effect is driven by the hydrolysis of ATP. This DNA-modified
electrochemical assay therefore reports on the enzymatic activity of SaXPD and likely
reflects better coupling of the 4Fe-4S cluster to the electrode during a helicase reaction. This
could have important implications for the function of XPD and coordination of nucleotide
excision repair within the cell. If during enzymatic activity, the efficiency of DNA-mediated
electronic signaling is increased, this could be used as a means for XPD to communicate its
activity to proteins downstream of XPD that contain 4Fe-4S clusters.
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Since SaXPD shares a midpoint redox potential with MutY and EndoIII, chemically, XPD
could shuttle electrons to and from MutY and EndoIII
in vitro
. AFM was used to test
directly if SaXPD can thus use DNA CT to signal to EndoIII and to localize to sites of DNA
damage (Figure 7). When SaXPD is incubated with samples of well-matched DNA and
DNA containing a mismatch, the protein preferentially binds to strands of DNA with
damage
64
, analogous to the effect observed for
E. coli
EndoIII.
22
Again, a mismatch is not a
substrate for XPD. SaXPD L325V is a mutant that was found to be deficient in DNA CT
electrochemically, and here too, as found with EndoIII mutants, deficient in CT signaling,
the L325V mutant of SaXPD is unable to redistribute to sites of damage. Significantly, when
mixtures of EndoIII and SaXPD are mixed with the DNAs, the proteins redistribute to sites
of damage, but when the mutants SaXPD L325V or EndoIII Y82A are swapped for their
WT counterparts in the mixtures, redistribution is no longer observed. These results provide
evidence that EndoIII and SaXPD can signal one another via DNA CT to localize to sites of
damage.
64
Indeed, even though EndoIII and SaXPD are from different organisms, even
different kingdoms, the fact that they contain 4Fe-4S clusters that share a similar redox
potential and can electrochemically couple to the DNA
π
-stack, allows them to use DNA CT
as a means of signaling, cooperating from a distance to find sites of DNA damage.
Cross-talk between repair pathways via DNA-mediated signaling in cells
DinG is a damage-inducible, ATP-dependent helicase from
E. coli
that also contains a
4Fe-4S cluster and shares significant homology to SaXPD.
65
–
67
DinG, like XPD, unwinds
DNA with a 5
′
to 3
′
polarity. Most substrates that contain a 5
′
to 3
′
overhang of at least 15
base pairs can be unwound by DinG, though DinG cannot unwind double-stranded DNA
(dsDNA). Uniquely, DinG can, however, unwind R-loops, which are RNA-DNA hybrids
that form within a bubble. R-loops have been shown to be one of the target substrates of
DinG
in vivo
.
68
Similar to EndoIII, the redox potential of the [4Fe-4S]
2+/1+
couple when not
bound to DNA was found to be ~−400 mV vs. NHE using titrations with redox mediators.
66
The electrochemical assay used to test the DNA-bound redox characteristics of EndoIII,
MutY, and SaXPD was used to examine DinG. It was shown that the DNA-bound potential
of DinG is ~ 80 mV vs. NHE.
50
Again, this is the same DNA-bound redox potential
observed for the glycosylases EndoIII and MutY, and the helicase SaXPD. It appears that
the emerging class of DNA-processing containing enzymes 4Fe-4S clusters all share a
DNA-bound redox potential, which could facilitate self-exchange electron transfer reactions
among the proteins. Based on the results for EndoIII,
20
,
21
we considered that the couple
observed when bound to DNA is the [4Fe-4S]
3+/2+
couple, shifted towards oxidation upon
binding to DNA. The substrate used to characterize DinG electrochemically was a 20-mer
duplex with a 15-mer 5
′
to 3
′
single-stranded overhang, a substrate that can be unwound by
DinG. When ATP is added to DinG on DNA-modified electrodes, a significant increase in
current is observed, similar to the effect observed for SaXPD (Figure 8). The increase in the
electrochemical signal over time for DinG is ~5–10 fold higher than that seen for SaXPD,
which has a lower rate of ATP hydrolysis, establishing a correlation between the rates of
ATP hydrolysis and increase in signal.
25
The AFM redistribution assay testing cooperative
DNA-mediated signaling between DNA-binding proteins containing 4Fe-4S clusters also
showed results analogous to that seen for SaXPD. DinG is found to redistribute to DNA
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containing a mismatch site that attenuates DNA CT. Moreover, DNA-mediated signaling
between EndoIII and DinG was also established, since redistribution to long strands of DNA
containing a mismatch are observed for mixtures of DinG and EndoIII, but not when the
CT-deficient EndoIII Y82A mutant is mixed with DinG. These
in vitro
experiments indicate
that DinG, EndoIII, and MutY are equipped to and may utilize DNA-mediated CT to
cooperatively and efficiently find damage.
The important question of whether or not DNA-mediated CT signaling occurs between
repair enzymes containing 4Fe-4S clusters from different pathways
in vivo
was next
examined. Since DinG is from
E. coli
, genetics experiments can be conducted to explore
whether cooperative signaling occurs among EndoIII, MutY, and DinG
in vivo
. Based upon
the AFM results for DinG and the proposed model for redistribution, one would expect that
the absence of DinG would lead to a decrease in MutY activity within cells, which was
measured using a
lac+
forward reversion assay in the CC104 strain of bacteria, as described
earlier.
57
,
58
When DinG is knocked out of CC104, an increase in the number of
lac
+
revertants is observed, reflecting the predicted decrease in MutY activity.
50
If it is the
4Fe-4S cluster of DinG and its participation in DNA-mediated signaling that is required for
MutY to fully function, then EndoIII, with its analogous 4Fe-4S cluster could be used to
rescue MutY activity. Complementation plasmids expressing various mutants of EndoIII
were used to test if the observed signaling between MutY and DinG was due to DNA-
mediated CT signaling. Indeed, expression of EndoIII D138A, which is catalytically inactive
but can bind DNA and is CT-proficient, restores MutY activity. Moreover, expression of the
CT-deficient EndoIII Y82A mutant does not rescue MutY activity.
50
Thus it appears that
DinG is assisting MutY in preventing GC:TA transversions via DNA-mediated CT, assisting
MutY in finding its target lesion, and substitution of EndoIII or MutY accomplishes the
same goal.
In a second genetic study, using a sensitive assay for DinG activity, we then asked the
question another way: If DinG can assist MutY or EndoIII, can EndoIII similarly assist
DinG? A strain of bacteria designated InvA, constructed so that the activity of DinG is
crucial, was used to show that EndoIII cooperates with DinG to dismantle R-loops. In
conditions that promote stalled replication forks resulting from the collision of the fork with
the transcription machinery, DinG is required for normal cell growth.
68
This requirement
has been attributed to DinG being required to unwind buildups of R-loops at these stalled
replication forks. A significant growth defect is observed when the gene encoding DinG is
knocked out of the InvA strain
68
, and overexpression of RNaseH, which degrades R-loops,
rescues the growth defect. To test if EndoIII cooperates with DinG, assisting DinG in
targeting and dismantling R-loops, the gene encoding EndoIII was silenced in InvA. When
EndoIII is knocked out of the InvA strain, a significant growth defect is observed (Figure
9).
50
This growth defect appears to be due to signaling between EndoIII and DinG, since
overexpression of RNaseH rescues the growth defect. But is this cooperation due to DNA-
mediated CT signaling? Complementation with a plasmid encoding EndoIII D138A, which
is catalytically inactive but CT-proficient, also rescues the growth defect, while, strikingly,
expression of the CT-deficient mutant EndoIII Y82A does not (Figure 9). These results in
combination with the results from the AFM redistribution assay and
lac
+
forward reversion
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assay strongly point to MutY, EndoIII, and DinG all utilizing DNA CT as a means of
cooperative signaling within cells to more efficiently repair DNA.
4Fe-4S clusters in Eukaryotes
While the work presented here has focused on DNA-processing enzymes that contain
4Fe-4S clusters in prokaryotes and archaea, 4Fe-4S clusters have also now been found in a
wide array of enzymes in eukaryotic organisms (Table 1). It is interesting to consider that
only a decade ago, few DNA-processing enzymes were known to contain 4Fe-4S clusters.
One reason the presence of iron-sulfur clusters in these enzymes is now being uncovered is
that isolating the cluster-loaded protein appears to depend greatly on the expression and
purification conditions. Less typical expression conditions, using low concentrations of
inducers of gene expression and carrying out the expression at low temperatures, have been
needed to obtain high yields of cluster-loaded proteins.
23
,
50
,
59
,
63
,
69
In other examples, such
as for yeast polymerase
δ
, the proteins must be purified from the native organism in order to
be loaded with a 4Fe-4S cluster.
70
Using these new techniques and upon closer examination,
many 4Fe-4S clusters are being found in an array of eukaryotic enzymes. The 4Fe-4S
clusters have been found in members of the nucleotide excision and base excision repair
families such as XPD, Ntg2, and OGG1, as well as in a host of helicases
59
–
61
,
71
,
72
. Enzymes
functioning in recombinatorial repair such as FancJ and DNA2 also contain 4Fe-4S clusters
in eukaryotes.
61
,
71
–
74
Even enzymes involved in the vital processes of transcription and
replication such as RNA polymerase, DNA polymerases
α
/
δ
/
ε
, and primase have now been
shown to contain 4Fe-4S clusters.
70
,
71
,
75
–
78
The role of the 4Fe-4S clusters in all of these
enzymes remains to be established Yet it is clear that evolution has preserved and even
expanded the presence of the 4Fe-4S cluster across the phylogeny. Mutations in enzymes
containing 4Fe-4S clusters are, moreover, linked to human disease such as photosensitivity
syndromes and early onset breast cancer.
59
,
61
Furthermore, disruption of the iron-sulfur
cluster biogenesis machinery in yeast has been directly linked to genomic instability.
79
–
81
It
is clear, then, that these clusters play an important role within these enzymes. Based upon
the data we have presented, one may consider that one role for these clusters may be to help
coordinate DNA repair.
One issue that has not been extensively studied is whether the oxidation state of the 4Fe-4S
cluster serves to modulate the catalytic activity of a given protein in this family of proteins.
Experiments to test activity of DNA-processing enzymes containing 4Fe-4S clusters in their
oxidized versus reduced forms are now required. It is tempting to propose that these clusters
may serve to facilitate DNA-mediated CT signaling in order to coordinate not only DNA
repair, but also transcription and replication in eukaryotes. Can redox signaling from a
distance serve to turn on replication, or perhaps more functionally, turn off replication by
oxidizing the 4Fe-4S cluster of DNA polymerases under conditions of oxidative stress?
Replication and transcription requires an extensive degree of coordination is at the
replication fork or site of transcription, and involves many steps carried out by elaborate
complexes of numerous enzymes. Utilizing DNA as a means of communication would serve
as an attractive mechanism to help facilitate this critical process.
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Conclusion
DNA CT chemistry thus offers a means to carry out redox chemistry at a distance and in a
fashion that depends upon the integrity of the intervening base pair stack. We have seen how
this chemistry may be utilized as a sensor of oxidative stress, both in funneling damage and
in oxidizing DNA-bound protein sensors. We have also seen how proteins containing
4Fe-4S clusters may signal one another using DNA CT chemistry and how specifically this
signaling may be used as a first step in efficiently redistributing repair proteins to sites of
DNA lesions for repair. The association of 4Fe-4S clusters, common redox cofactors in
biology, with proteins involved in DNA repair is being found with increasing frequency and
has apparently been preserved in higher organisms that carry out still more complex modes
of repair. Utilizing DNA CT with these redox cofactors provides a role for these clusters in
the repair proteins along with a mechanism to understand how the cell achieves the high
level of efficient repair we require. Indeed, given findings of 4Fe-4S clusters now in proteins
involved in all aspects of DNA processing, it is tempting to suggest that here too these redox
cofactors may be present to carry out DNA CT. Thus DNA CT could provide a general
means of signaling among DNA-bound proteins across the genome. Certainly a range of
experiments to explore the many ways this redox chemistry may be utilized within the cell is
now required.
Acknowledgments
Funding Statement: Research funded by the Center for Chemical Signaling through the Gordon and Betty Moore
Foundation, and the National Institutes of Health (GM61077).
We are grateful to our many coworkers and collaborators for their diligence and creativity in exploring this
chemistry.
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Figure 1.
Measuring DNA CT with metal complexes. Shown is a characteristic DNA assembly used
to monitor DNA-mediated redox chemistry in solution, using metallointercalators to monitor
luminescence quenching by electron transfer through the DNA base stack.
6
The chemical
structures of the covalent donor and acceptor, Ru(phen)
2
dppz
2+
(phen = 1,10-
phenanthroline, dppz = dipyrido[3,2-
a
:2
′
,3
′
-
c
]phenazine) (left) and Rh(phi)
2
phen
3+
(phi =
9,10-phenanthrenequinone diimine) (right), respectively, are shown.
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Figure 2.
Measuring DNA CT in single molecules. A single DNA duplex is made to covalently bridge
a gap in an electronically wired, carbon nanotube device such that the measured current flow
through the device reflects DNA CT efficiency.
10
To confirm that charge flow through the
device is DNA-mediated and to illustrate the sensitivity of DNA CT to single base
mismatches, an experiment was designed to allow the introduction of a single base
mismatch through thermal dehybridization and rehybridization of the bridging duplex (left).
One strand of the DNA duplex is covalently attached at either side of the gap (black), while
the other, noncovalent strand is cycled between a well matched strand (turquoise) and
strands with a single base mismatch (orange, purple) by sequential dehybridization, rinsing,
and rehybridization. During this cycling between well matched and mismatched duplexes,
the source-drain current (
I
SD
) for the device was measured at a constant gating voltage (
V
G
= −3V) and plotted for each bridging duplex (right plot, where the colors and numbers of the
duplexes in the left illustration correspond to those on the plot). This experiment clearly
illustrates the high sensitivity of DNA CT to single base mismatches; DNA CT is inhibited
when the device is bridged with a mismatched duplex and restored when the device is
rehybridized with a well matched duplex.
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