of 34
Redox Chemistry in the Genome: Emergence of the [4Fe4S]
Cofactor in DNA Repair and Replication
Jacqueline K. Barton
,
Rebekah M. B. Silva
, and
Elizabeth O’Brien
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena CA
91125
Abstract
Many DNA-processing enzymes have been shown to contain a [4Fe4S] cluster, a common redox
cofactor in biology. We find using DNA electrochemistry that binding of the DNA polyanion
promotes a negative shift in [4Fe4S] cluster potential, which corresponds thermodynamically to ~
500-fold increase in DNA binding affinity for the oxidized [4Fe4S]
3+
cluster versus the reduced
[4Fe4S]
2+
cluster. This redox switch can be activated from a distance using DNA charge transport
chemistry. DNA-processing proteins containing the [4Fe4S] cluster are enumerated with possible
roles for the redox switch, highlighted. A model is described where repair proteins may signal one
another using DNA-mediated charge transport as a first step in their search for lesions. The redox
switch in eukaryotic DNA primases appears to regulate polymerase handoff, and in DNA
polymerase
δ
, the redox switch provides a means to modulate replication in response to oxidative
stress. Thus we describe redox signaling interactions of DNA-processing [4Fe4S] enzymes as well
as the most interesting potential players to consider in delineating new DNA-mediated redox
signaling networks.
Keywords
DNA charge transport; iron sulfur clusters; base excision repair; DNA primase; DNA polymerase;
redox signaling; oxidative stress
I. Introduction
Iron sulfur clusters are modular, tunable metal cofactors found in all domains of life that
serve as one-electron carriers operating over a wide range of physiological potentials, from
approximately −500 mV vs. NHE to 300 mV vs. NHE (
1
3
). Cytosolic and membrane-
bound proteins have been found to coordinate a cubane [4Fe4S] cluster at a range of redox
potentials that vary depending on the local protein environment and solvent exposure (Figure
1) (
4
,
5
). Found in the [4Fe4S]
2+
resting state, high potential [4Fe4S] clusters, like those in
high potential iron (HiPIP) proteins, can be oxidized to the [4Fe4S]
3+
state and lower
potential clusters, like those in ferredoxins, can be reduced to the [4Fe4S]
+
state (
6
). These
cofactors commonly mediate redox reactions in nitrogen fixation, photosynthesis, and
respiration (
7
9
), often acting in a chain of metal cofactors within an otherwise insulating
protein matrix (
10
,
11
).
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Thirty years ago, these [4Fe4S] clusters were found also to be associated with a protein
involved in DNA repair (
12
), and over time, more and more proteins involved in DNA
processing were found to contain [4Fe4S] clusters. What were their roles? Were they
structural factors or perhaps ancestral relics? As described here, we are finding that these
[4Fe4S] clusters carry out redox reactions in DNA-processing proteins, serving as redox
switches to regulate binding to the DNA polyanion.
[4Fe4S] Cluster Biogenesis and Loading to Target Proteins
After decades of progress, it is now understood that the incorporation of [4Fe4S] cofactors
occurs through a highly regulated and coordinated series of metabolically expensive steps by
a network of iron sulfur cluster biogenesis proteins (
13
19
). General biogenesis is carried
out by the ISC pathway (Figure 2), where free iron and reduced sulfide (S
2-
, derived from
free cysteine) are scaffolded onto components of biogenesis machinery, delivered, and
loaded to apo-protein targets in a process facilitated by chaperone proteins and driven by
ATP hydrolysis. In prokaryotes, this entire process occurs within the cytosol. In eukaryotes,
biogenesis begins in the mitochondria, and for cytoplasmic and nuclear-bound cluster
proteins (which include repair and replication enzymes), is completed in the cytoplasm by
the cytosolic iron sulfur assembly (CIA) machinery (
16
). At present, specialized biogenesis
components have not been identified for [4Fe4S] repair proteins in prokaryotes. However,
prokaryotic biogenesis has been linked to pathogenesis and antibiotic resistance (
20
). Future
studies will likely be quite informative for uncovering new regulatory roles for FeS
biogenesis in all domains of life.
Mechanisms of protein target recognition by biogenesis machinery have been brought to the
forefront recently. Bioinformatic signatures of the coordinating cysteines in repair and
replication proteins are surprisingly weak (
21
); however, newly discovered target sequences
recognized by biogenesis machinery, which include an LYR motif and a KKX
6–10
KK
sequence, have been found to be essential for association with an ISC co-chaperone in yeast
(
13
). Continued investigation of recognition motifs will be important for understanding
cluster biogenesis, unraveling new facets in iron metabolism, and identifying new cluster
proteins, such as those involved in DNA processes.
DNA-Processing [4Fe4S] Enzymes
The surprising discovery of a [4Fe4S] cluster in the base excision repair (BER) glycosylase
Endonuclease III (EndoIII) from
Escherichia coli
(
E. coli
) in 1989 (
12
) soon led to the
discovery of [4Fe4S] clusters in MutY (an EndoIII paralog) and Uracil DNA Glycosylase in
Archaeoglobus fulgidus
(AfUDG) (
22
,
23
). Over the following decades, nucleic acid
processing enzymes across several pathways were shown to contain [4Fe4S] cofactors
(Table 1) (
24
30
). In most cases, discovery of the [4Fe4S] cluster occurred years after the
first isolation of the gene products (
27
). As predictive tools and protein isolation methods
become more and more sophisticated, we and others expect that even more [4Fe4S] clusters
will be observed in essential DNA processing enzymes (
27
,
31
).
The question of what role the [4Fe4S] clusters played, however, was less straightforward to
answer, as early studies demonstrated that the clusters were isolated in the electron
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paramagnetic resonance (EPR) silent [4Fe4S]
2+
state and resistant to powerful chemical
oxidants and reductants (
12
,
23
,
32
). Moreover, the spectroscopic signature of coordinating
cysteines was unchanged upon binding a damaged substrate, leading to the initial conclusion
that the cluster had a structural role (
12
,
33
,
34
). MutY, however, could be denatured and
refolded in the apo form, challenging this early conclusion (
35
). A substrate-sensing role
was proposed for the cluster in light of this result, but a general, chemical function for the
cofactor eluded observation.
II. Characterizing the Fundamental Properties of DNA-Mediated Charge
Transport
In addition to the metabolic expense undertaken by cells to load a cluster into a target
protein, placing an iron-containing cofactor in a DNA-binding enzyme can put the bound
nucleic acid at risk of damage. A labile ferrous iron from the cofactor can undergo Fenton
chemistry, creating reactive oxygen species and damaging nearby DNA bases (Figure 1)
(
36
). Why then does Nature spend the requisite energy incorporating a redox-active
inorganic cofactor into a DNA-processing enzyme?
At the same time that these proteins involved in DNA processing were being found to
contain [4Fe4S] clusters, experiments were being conducted to characterize DNA charge
transport chemistry (DNA CT), where electrons rapidly migrate through well stacked duplex
DNA (
37
). The native substrate of these [4Fe4S] enzymes, double stranded DNA (dsDNA),
was initially predicted to conduct charge in the dry, solid state (
38
), as the
π
-stacked DNA
bases resemble the structure of graphite, a conductive material (Figure 3). To assess whether
DNA conducted charge in biologically relevant aqueous conditions, new platforms were
developed to examine this chemistry. Two important characteristics of this chemistry
emerged: (i) DNA CT can occur over long molecular distances with shallow distance
dependence, and (ii) DNA CT is exquisitely sensitive to perturbations in
π
-stacking of the
bases.
A range of studies using DNA-bound electron donors and acceptors were used to
characterize DNA CT chemistry. In an early photophysical study, a DNA oligomer was
prepared containing a tethered luminescent ruthenium intercalator at one end and an
intercalating rhodium oxidant at the other. While the tethered, DNA-bound ruthenium
complex luminesced in the absence of the rhodium complex, in its presence, the
luminescence of the ruthenium complex was quenched by electron transfer, remarkably over
a distance > 40 Å (
39
). In a subsequent experiment using ethidium as the luminescent donor,
electron transfer quenching was also evident but was attenuated in the presence of a single
base mismatch intervening between the bound ethidium and rhodium (
40
). Long-range CT
through a 63 bp duplex DNA substrate has been observed with DNA-intercalating
photooxidants, where the DNA-bound photooxidant can promote oxidative damage at
guanine residues from a distance. A covalent rhodium photooxidant at one end of a DNA
duplex, for example, oxidizes guanine bases at the 5
- guanine of a guanine doublet, the site
of low oxidation potential, through DNA CT, generating 7,8-dihydro-8-oxo-2
-
deoxyguanosine lesions 200 Å from the site of intercalation (
41
). Experiments monitoring
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base-base CT utilizing 2-aminopurine furthermore showed that these DNA CT reactions can
occur on the picosecond timescale, gated by the motions of the bases (
42
), and moving 10
times the single-step tunneling distance through protein in a miniscule fraction of the time
(
10
,
11
,
37
,
43
,
44
)!
Again, however, perturbations in base stacking, as occur with base mismatches or kinks in
the DNA, turn off this long range CT chemistry. In fact, proteins that bind and kink the
DNA, such as TATA-binding protein, can be used to turn off DNA CT. In contrast, proteins
that bind DNA without affecting DNA base stacking, as with histones, do not alter DNA CT
(
45
47
).
We also explored DNA CT in the ground state using DNA electrochemistry. Here, as with
the photophysical studies, we observe long range CT as long as the DNA is well stacked.
Indeed, ground state CT through DNA was observed over 100 base pairs to a tethered,
intercalating redox probe, methylene blue, using DNA-modified gold electrodes, but a single
base mismatch in the 100-mer was sufficient to attenuate the redox signal severely (
48
).
III. Measuring Redox Potentials of [4Fe4S] Enzymes Bound to DNA
For our early electrochemistry studies, we had used proteins to modulate DNA CT to a small
DNA-bound redox probe (
46
), but we considered that DNA electrochemistry could be used
also to examine DNA CT to a redox cofactor
within
a DNA-bound protein. Could a DNA-
binding protein containing a redox cofactor carry out DNA CT chemistry? If so, DNA CT
experiments could be used to characterize the redox centers of DNA-binding proteins and to
determine their DNA-bound potentials.
DNA-Mediated Electrochemistry
DNA-modified Au electrodes have become a useful platform for assessing whether a DNA-
processing, [4Fe4S] enzyme is redox-active in solution under physiologically relevant
conditions (Figure 4). Gold surfaces are functionalized with alkanethiol-modified DNA
duplexes through formation of a thiol-gold bond. The Au can be used as the working
electrode in a three-electrode cell after surface washing and passivation (
48
50
). DNA-
bound redox potentials of [4Fe4S] proteins can be measured with this method, where charge
flows from the electrode through the DNA to the cluster. In this platform, the DNA,
functionalized onto an electrode surface, is biologically accessible; restriction enzymes, for
example, can cut the DNA on the electrode with sequence specificity, as in solution.
Electrochemical studies on these platforms have been central to the prediction and discovery
of redox signaling between DNA-bound [4Fe4S] enzymes across different repair pathways
(
51
53
).
We first examined MutY, EndoIII, and AfUDG, the three base excision repair proteins found
to contain [4Fe4S] clusters (
32
). Earlier studies using strong chemical oxidants and
reductants had suggested that the clusters were redox-inactive at physiological potentials,
but these studies had been conducted in the
absence
of DNA. It was reasonable to consider
that binding the DNA polyanion might change the potential of the cluster within the protein.
Our studies showed first that a reversible signal was detectable at ~80 mV versus NHE,
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within the physiological window, for each of these proteins bound to the DNA-modified
electrode. The potentials were consistent with those found for the clusters in HiPIP proteins
and were at equal potentials for all of the repair proteins examined. Moreover, the presence
of an abasic site on the DNA intervening between the bound protein and the electrode
surface served to attenuate the signal from the cluster. This result established two important
points: (i) we were measuring the
DNA-bound
potential of the protein and (ii) we were
observing DNA-mediated CT between the electrode and the cluster within the protein. Many
repair and replication proteins have now been studied using this DNA electrochemistry
platform, and in each case we have observed reversible, redox signals in the physiological
potential range, near ~80 mV vs. NHE, corresponding to cycling between the [4Fe4S]
2+
and
[4Fe4S]
3+
oxidation states (
28
,
32
,
51
,
52
,
54
57
).
It is noteworthy that the most recent generation of DNA-modified electrodes utilizes a
multiplexed chip so multiple experimental conditions can be examined in parallel (Figure 4)
(
48
50
). Patterned, silicon chips with sixteen independently addressable Au electrodes
uniform in area can be physically divided into four quadrants and used to monitor the redox
activity of a single protein on as many as four different DNA substrates on the same surface.
The platform can also be used to compare CT efficiency of WT and mutant protein on the
same chip. All of the experiments described can moreover be carried out under strictly
anaerobic conditions.
Graphite Electrochemistry to Compare DNA-bound and DNA-free [4Fe4S] Cluster
Potentials
We became interested in monitoring directly the shift in potential for [4Fe4S] repair proteins
associated with binding DNA, and that required measuring the protein potential both in the
absence and presence of DNA. DNA electrochemistry on Au electrode surfaces is amenable
to measuring physiological potentials ranging from −200 mV to +500 mV vs. NHE (
28
,
50
59
). However, scanning beyond this range is necessary to observe a redox signal from a
protein in the absence of DNA, which has a higher (more reductive) midpoint potential due
to the absence of the DNA polyanion (
51
,
53
,
56
,
60
,
61
). In order to measure the DNA-free
and DNA-bound redox potentials of EndoIII, highly oriented pyrolytic graphite (HOPG)
electrodes were used (
60
). Bare surfaces or surfaces modified with pyrene-functionalized
duplex DNA (which creates a DNA monolayer through
π
stacking between pyrene and
graphite) facilitated direct comparison of DNA-free and DNA-bound EndoIII [4Fe4S]
cluster redox potentials. A negative shift of approximately 200 mV was observed for the
DNA-bound EndoIII relative to DNA-free EndoIII. Given that protein binding importantly
does not lead to a large conformational change in the protein or DNA, this shift in redox
potential for the [4Fe4S] cluster thermodynamically reflects a roughly 500-fold tighter DNA
binding affinity for the oxidized [4Fe4S]
3+
state, relative to the reduced [4Fe4S]
2+
state,
based on the Nernst equation (Figure 5). Oxidation of the [4Fe4S]
2+
cluster to the [4Fe4S]
3+
state thus should serve as a redox switch for DNA binding.
Spectroscopic Observation of [4Fe4S] Cluster Redox Activity in DNA-Processing Proteins
Electrochemical observations of the [4Fe4S] clusters in DNA-processing enzymes was also
complemented by spectroscopic analysis. EPR spectroscopy requires chemical or
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electrochemical conversion of the resting, diamagnetic [4Fe4S]
2+
enzymes to the
paramagnetic oxidized [4Fe4S]
3+
or reduced [4Fe4S]
+
redox states, and thus was used to
establish the resting state for the DNA-bound repair protein as [4Fe4S]
2+
(
28
,
62
65
). We
also demonstrated that the DNA-bound protein can be oxidized photochemically, from a
distance, using DNA CT from a distantly tethered intercalating photooxidant (
32
,
51
).
Importantly, we were also able to demonstrate spectroscopically that the cluster could be
oxidized by guanine radicals, generated using flash-quench experiments monitored by
transient absorption spectroscopy (
66
). Indeed, these studies highlight how the oxidized
[4Fe4S]
3+
cluster could be generated within the cell,
from a distance
using DNA CT from
guanine radicals under conditions of oxidative stress, and in so doing, activating the DNA
repair machinery.
IV. A Shift in Cluster Potential Reflects a Redox Switch in DNA Binding
While we had seen several examples of DNA binding yielding a shift in redox potential for
the clusters within the repair proteins, from which one can infer a difference in DNA binding
affinity for the protein with a [4Fe4S]
3+
cluster versus a [4Fe4S]
2+
cluster, we were not at
first able to measure this difference directly. In the absence of DNA, the [4Fe4S]
3+
cluster
degrades further to a [3Fe4S]
+
cluster, which affects protein binding. As a result, binding
affinities for the [4Fe4S]
3+/2+
clusters needed to be determined anaerobically.
Recently we found that microscale thermophoresis could be used under anaerobic conditions
to carry out DNA binding experiments for the [4Fe4S] proteins in the two oxidation states.
EndoIII containing the [4Fe4S]
3+
cluster was first generated on DNA-modified electrodes
and then, under strictly anaerobic conditions, the thermophoresis experiments were
conducted (
53
). Consistent with the shift in potential associated with DNA binding, oxidized
EndoIII with the [4Fe4S]
3+
cluster was indeed found to bind >500 times more tightly to
dsDNA than the reduced EndoIII with the [4Fe4S]
2+
cluster (Figure 5). This difference in
binding affinity is understandable based simply on electrostatic considerations. In fact,
calculations based upon the distance of the cluster to the DNA polyanion and the intervening
protein dielectric well reflect the change in binding affinity that we see. It is interesting in
that context that we find similar shifts in potential for all of the DNA repair proteins
examined, and the clusters generally appear to be ~ 20 Å from the polyanionic DNA
backbone. Based upon these results, then, we can consider that binding of these repair
proteins to the DNA polyanion serves to tune the potential of the cluster by altering the
electrostatic environment, activating the cluster toward oxidation and lowering the
[4Fe4S]
3+/2+
couple into a physiologically accessible potential range.
V. [4Fe4S] Proteins in Nucleic Acid Processing and Repair
Thousands of DNA damage sites are generated by endogenous and exogenous agents in each
cell daily (
67
,
68
). An arsenal of DNA repair pathways have evolved to address the
structurally and chemically diverse lesions, though a comprehensive understanding of how
repair pathways efficiently identify and remove damaged bases has remained elusive (
69
,
70
). In the case of the repair proteins that contain [4Fe4S] clusters, what they share is a low
copy number within the cell and a moderate specificity in binding their target lesion versus
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unmodified DNA. But with few players and not a high specificity in targeting, how do they
effectively find all the lesions within the cell on a timescale appropriate to the organism?
We have considered that DNA charge transport chemistry might provide a first step in
localizing repair proteins that contain these clusters in the vicinity of lesions, essentially
redistributing the proteins to regions of damage, irrespective of the actual lesion
characteristics as long as the damage interferes with DNA CT. We had actually found earlier
that many common base lesions interfere with DNA CT (
71
). Moreover, the fact that these
proteins share a similar redox potential means that essentially they can work together,
transferring electrons from one to another to carry out a first scan of the genome.
A Model for Scanning the Genome Utilizing DNA CT Chemistry
To describe how DNA-mediated CT chemistry could be utilized for more efficient lesion
detection, we developed a model for redox signaling among a network of [4Fe4S] repair
proteins (Figure 6) (
32
). Upon association of freely-diffusing protein in the [4Fe4S]
2+
state
onto duplex DNA, the protein is activated toward oxidation and can reduce a distally bound
protein from the [4Fe4S]
3+
state to the more weakly binding [4Fe4S]
2+
state (Figure 5, top).
Alternatively, guanine radicals (G
.+
), a product of oxidative stress, can generate the
[4Fe4S]
3+
species by DNA CT over long molecular distances (
41
,
66
,
72
). Iterations of this
scanning can occur throughout the cell. However, if there is an intervening region of dsDNA
containing a lesion that attenuates CT, redox signaling between the cluster proteins is
interrupted. Both proteins in the tightly-binding [4Fe4S]
3+
state persist on the DNA and can
then localize to the precise site of damage (Figure 5, bottom).
We have, by now, found many types of DNA repair proteins containing [4Fe4S] clusters that
are involved in redox signaling on DNA, and we have probed how these redox signaling
networks work cooperatively utilizing DNA CT through their [4Fe4S] clusters. We have also
found CT deficiencies in mutated proteins strongly linked to disease. In the context of redox
signaling, included below is an overview of the major repair protein families that coordinate
a [4Fe4S] cluster, and illustrations of how they may utilize redox signaling.
Glycosylases
Glycosylases are key players in BER, a highly conserved pathway responsible for
recognizing and removing single-base lesions generated by spontaneous deamination,
alkylating agents, and oxidative stress, among other sources of damage (Figure 7) (
73
,
74
).
For EndoIII, MutY, and AfUDG, biochemical studies have very elegantly demonstrated that
mutations at coordinating cysteines or in the cluster binding domain can affect protein
expression, enzymatic function, and DNA binding, even though the cluster is located
remotely from the glycosylase active site and there is not a large conformational change
associated with the binding to DNA (
34
,
75
77
). Specific to mammalian BER, a connection
has been established between the glycosylases, NTHL1 and MUTYH, and multiple cancers,
most notably MUTYH-associated polyposis (MAP). These syndromes are characterized by
increased risk of aggressive, early-onset colorectal cancer (
68
,
74
,
78
).
As described above, several BER glycosylases were demonstrated to participate in DNA-
mediated CT chemistry (
32
). Further examination of the available structures of EndoIII in
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the free and DNA-bound forms revealed a pathway of aromatic residues between the cluster
and the DNA, separated by 15-20 Å, and predicted to provide a CT pathway (
10
,
11
,
79
,
80
).
Informed by these crystal structures, several key mutants of EndoIII have been generated
and characterized, including a CT deficient/enzymatically proficient mutant Y82A, a
complementary CT proficient but enzymatically deficient mutant D138A, and charge-flipped
mutants to explore the electrostatics near the cluster (
61
,
79
,
80
). Unique to the HiPIP-like
[4Fe4S] repair proteins, the DNA polyanion is the governing factor that shifts the potential
and stabilizes the cluster in the [4Fe4S]
3+
state (
5
,
61
). Furthermore, redox sensing of base
stacking perturbations on DNA-modified gold electrodes containing a lesion has been
observed, even when that lesion is not a substrate for the glycosylase (
32
,
80
).
Superfamily 2 (SF2) 5'
3' Helicases
SF2 helicases were the second family of enzymes discovered to coordinate a [4Fe4S] cluster
(
65
). SF2 helicases are NTP-dependent proteins that directionally translocate and unwind
duplexes (
81
). Distinct from the glycosylase family, the FeS helicases are involved in several
repair pathways (Figure 7). These pathways respond to stress from multiple sources, and
there are many examples of extensive crosstalk and cooperativity among a complex network
of repair pathways that include SF2 helicases with [4Fe4S] clusters (
27
). As might be
expected, mutations in SF2 helicases containing [4Fe4S] clusters are associated with a host
of genetic disorders and cancers and are being targeted for cancer therapies (
82
,
83
).
A common theme with SF2 helicases is the multifunctional nature of their activities within
the cell (Figure 7). In bacteria, DinG resolves R-loops, RNA:DNA hybrids formed at
collisions between replication and transcription machinery; DinG has also been shown to be
active on D-loops (displacement loops, triple stranded DNA) (
64
,
84
). Notably, cysteine
mutants of DinG are more susceptible to proteolytic degradation
in vitro
(
64
). A new, well-
conserved bacterial protein YoaA, was identified in a genetics screen to be involved in
coordinating repair and replication machinery at blocked replication forks. Based on
sequence similarity to DinG, YoaA was predicted to be a [4Fe4S] protein (
85
).
The first archaeal/eukaryotic [4Fe4S] SF2 helicase discovered, XPD, is part of the
transcription factor II H (TFIIH) complex and is involved in both nucleotide excision repair
(NER) and transcription initiation (
65
). In NER, helicase activity is critical for removing
damaged oligomers, which can be disrupted by mutating coordinating cysteines. In contrast,
only the association of XPD with TFIIH is needed to initiate transcription, which is thought
to aid assembly of other proteins with TFIIH. Many other facets of the XPD have also been
studied, including regulation of XPD by other proteins, the timing of cluster insertion
relative to XPD incorporation with TFIIH, how XPD is involved in cell cycle control, and a
role for XPD in preventing oxidative damage in the mitochondria (
86
).
Both XPD and DinG can participate in DNA CT chemistry with a shared DNA-bound redox
potential of approximately 80 mV vs. NHE and sense signal attenuating lesions (
54
,
59
).
Further probing of the CT activity found that upon addition of ATP, the signal for XPD and
DinG quite stunningly increases in a concentration-dependent manner without any shift in
the midpoint potential. Helicase activity thus increases the electrochemical signal through
better coupling of the cluster to the
π
-stacked DNA bases, essentially signaling the helicase
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activity through DNA CT. The increased coupling upon cofactor binding appears to be an
important feature of the SF2 helicase family that helps coordinate repair and replication, and
signaling from a distance that the helicase is active.
Another eukaryotic SF2 helicase, FANCJ has a role in several pathways; compromised
FANCJ function, which can result from mutations in the cluster binding domain, has been
linked to several cancers, and FANCJ upregulation has been found in many tumor types
(
87
). FANCJ is known to act on many substrates, including duplex substrates, D-loops and
G4 quadruplexes (
87
). FANCJ association with numerous other proteins, including BRCA1,
can depend on the timing of the damage response and the type of lesions generated.
Furthermore, FANCJ activity has been observed to alleviate replication stress through
resolution of stalled replication forks, and particularly at telomeres rich in G4 quadruplexes.
Two other SF2 helicases that coordinate a [4Fe4S] cluster, RTEL1 and Chlr1, resolve several
different types of structures in the process of facilitating replication (
83
,
88
). Both RTEL1
and Chlr1 have been also found to associate with various replication proteins. Mutations in
these enzymes are similarly associated with an array of diseases (
88
,
89
). Electrochemical
studies of these proteins have not yet been conducted, but studying these proteins in the
context of redox signaling will likely illuminate how these multifunctional enzymes may
coordinate their activities.
Helicase-Nucleases
In 2009, the first helicase-nuclease containing a [4Fe4S] cluster identified was AddB, part of
the AddAB heterodimer found in gram positive bacteria and some proteobacteria (
90
,
91
).
Helicase-nuclease activity is involved in double strand break (DSB) repair, which can be
caused by several factors, including collapsed replication forks. Located in the C-terminal
nuclease domain of AddB, the cluster was found to be essential for binding of DNA
substrates, but not essential for complexation with AddA or for maintaining structure,
though a stabilizing role of the cluster was suggested. The crystal structure of the AddAB in
complex with a DNA substrate revealed a DNA binding loop supported by the cluster
domain, providing explanation of why mutating coordinating cysteines abolished substrate
binding (
92
). A homologous [4Fe4S] helicase-nuclease, Dna2, was later found in eukaryotes
(
vide infra
).
Excision Nucleases
UvrC, an excision nuclease from
E. coli
, was reported in 2018 to be a [4Fe4S] protein (
28
).
Only found in bacterial and some archaeal NER pathways, UvrC (in complex with another
NER protein, UvrB) uniquely catalyzes incisions in the phosphodiester backbone both 5
-
and 3
- to damaged substrates (
93
). Distinct from the other known [4Fe4S] repair proteins in
bacteria, UvrC was found to coordinate an O
2
-sensitive [4Fe4S] cluster, causing protein
aggregation upon oxidative degradation. Mutation of coordinating cysteines to alanine led to
aggregation or severely reduced overexpression, the latter of which has been reported before
for MutY (
94
). Additionally, UvrC in its holo form binds to duplex DNA without lesions,
enabling demonstration that UvrC participates in DNA CT chemistry. UvrC shares a DNA-
bound midpoint potential with EndoIII, MutY, and DinG. UvrC has been noted in several
reports to be unstable
in vitro
and the scarce
in vivo
(~10 copies/cell), so continued
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investigation should provide insights as to how chemically and structurally diverse lesions
are detected by UvrC in its holo form.
Monitoring Redox Signaling Between Repair Proteins and Repair Pathways
Given the multitude of these clusters, their similarity in redox potential and their diversity in
lesions targeted, we became interested in examining how the proteins might cooperate in
searching for DNA lesions. To study cooperative, redox communication between [4Fe4S]
proteins, a series of
in vitro
and
in vivo
assays were developed to monitor signaling
networks.
In vitro
, atomic force microscopy (AFM) has facilitated visualization of [4Fe4S]
repair protein distribution on well-matched (WM) DNA versus duplex DNA containing a
single base mismatch (MM) (
53
,
55
,
59
,
79
,
80
). Though strands containing a single,
engineered C:A mismatch are not native substrates for any of these repair enzymes studied,
preferential binding to the 3.8 kb long MM strands, expressed as a binding density ratio of
1.5 (Figure 8), has been seen for all CT-proficient repair proteins. Mixtures of [4Fe4S]
proteins from different repair pathways, and, remarkably, mixtures of cluster proteins from
distinct organisms also signal cooperatively one to another, localizing to damaged strands.
Two factors have been found to affect the efficiency of the damage search: (i) the CT
proficiency of a protein and (ii) the extent to which the protein population is oxidized.
Mixing CT-proficient and CT-deficient proteins (
eg.
EndoIII and Y82A) impairs localization
to MM strands; the binding density ratio is 1. Protein samples that are 99% oxidized
generated anaerobically using DNA electrochemistry also do not redistribute to MM strands,
likely due to the >500-fold increase in binding affinity. Redistribution of oxidized EndoIII,
however, can be restored with addition of reduced DinG. The same phenomenon was seen in
the reciprocal experiment with oxidized DinG and reduced EndoIII. These data underscore
that long-range redox signaling between [4Fe4S] enzymes is dependent on the shared DNA-
bound redox potential and CT proficiency of the protein. Furthermore, these experiments
highlight the ability of the proteins to signal one another over kilobase distances.
Complementary genetics assays were developed using strains of
E. coli
that depend on the
repair activity of MutY, DinG, or UvrC for growth. Putative redox signaling networks can be
disrupted by genetically knocking out a signaling partner (
57
,
59
,
79
). Limited growth in the
knocked out strains therefore points to diminished repair activity occurring, owing to limited
availability of signaling partners (Figure 9). Complementation plasmids for EndoIII, which
include WT enzyme, Y82A, and D138A have been used to monitor how rescue with wild
type and EndoIII mutants can restore survival. With this genetic approach, evidence of
signaling among BER enzymes, DinG, and UvrC has been found to be necessary for repair
activity and growth of cells (
57
,
59
,
79
). Rescue with the CT competent but enzymatically
deficient D138A but not the CT deficient but enzymatically competent Y82A restores
growth in EndoIII knockouts, demonstrating that CT proficiency and not enzymatic activity
is needed for redox signaling and efficient repair activity in other pathways.
Diseases and Cancers Related to Mutations in [4Fe4S] Proteins
Mutations linking cancer with compromised repair activity by human [4Fe4S] proteins have
been reported (
24
,
58
). Investigations into the redox chemistry of mutant proteins has helped
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