of 21
The Oxidation State of [4Fe4S] Clusters Modulates the DNA-
Binding Affinity of DNA Repair Proteins
Edmund C. M. Tse
,
Theodore J. Zwang
, and
Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California 91125, United States
Abstract
A central question important to understanding DNA repair is how certain proteins are able to
search for, detect, and fix DNA damage on a biologically relevant timescale. A feature of many
base excision repair proteins is that they contain [4Fe4S] clusters that may aid their search for
lesions. In this report, we establish the importance of the oxidation state of the redox-active
[4Fe4S] cluster in the DNA damage detection process. We utilize DNA-modified electrodes to
generate repair proteins with [4Fe4S] clusters in the 2+ and 3+ states by bulk electrolysis under an
O
2
-free atmosphere. Anaerobic microscale thermophoresis results indicate that proteins carrying
[4Fe4S]
3+
clusters bind to DNA 550 times more tightly than those with [4Fe4S]
2+
clusters. The
measured increase in DNA-binding affinity matches the calculated affinity change associated with
the redox potential shift observed for [4Fe4S] cluster proteins upon binding to DNA. We further
devise an electrostatic model that shows this change in DNA-binding affinity of these proteins can
be fully explained by the differences in electrostatic interactions between DNA and the [4Fe4S]
cluster in the reduced versus oxidized state. We then utilize atomic force microscopy (AFM) to
demonstrate that the redox state of the [4Fe4S] clusters regulates the ability of two DNA repair
proteins, Endonuclease III and DinG, to bind preferentially to DNA duplexes containing a single
site of DNA damage (here a base mismatch) which inhibits DNA charge transport. Together, these
results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge
transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated
change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in
the lesion detection process.
Graphical Abstract
(top) DNA-mediated charge transport signaling is central to the first step of DNA damage
detection and occurs only through well-matched but not mismatched (denoted as a red box) DNA
duplexes between repair proteins (green and gray) carrying oxidized (orange) and reduced (purple)
[4Fe4S] metallocofactors. (bottom) Upon the oxidation of the [4Fe4S] cluster from 2+ to 3+, the
DNA binding affinity of the protein increases.
*
Corresponding Author: jkbarton@caltech.edu.
Notes
The authors declare no competing financial interest.
Supporting Information
The Supporting Information is available free of charge on the ACS Publication website.
Supplementary Figures (Figures S1–13), Supplementary Tables (Tables S1–9), and Supplementary Experimental Procedures
HHS Public Access
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Published in final edited form as:
J Am Chem Soc
. 2017 September 13; 139(36): 12784–12792. doi:10.1021/jacs.7b07230.
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INTRODUCTION
Exogenous and endogenous damage to deoxyribonucleic acid (DNA), originating from
sources such as reactive oxygen species, UV irradiation, and environmental mutagens,
causes lesions, mismatches, and base pair modifications in the genome of organisms on the
order of millions per cell per day.
1
,
2
Many repair mechanisms have evolved to uphold
genome integrity by repairing DNA damage caused by oxidative stress.
3
,
4
DNA repair
systems utilize diverse teams of proteins to respond, search, and repair DNA damage in a
timely fashion.
5
,
6
An increasing number of DNA-processing enzymes, including many that
are involved in the repair of DNA damage, have been found in archaea, bacteria, and
eukaryotes to contain [4Fe4S] clusters.
7
10
Elucidating the role of these clusters remains an
active area of investigation.
11
14
Initially, these clusters were proposed to serve primarily a
role in maintaining protein structural integrity. However, recent experiments demonstrate
that the presence of the [4Fe4S] cluster is not essential for the overall protein structure and
thermal stability of a DNA repair enzyme, even though the cluster contributes significantly
to DNA binding and enzymatic activity.
15
Other studies detailing processes loading iron-
sulfur clusters into repair proteins indicate that these multistep assembling procedures are
resource-intensive and suggest that these clusters may have other roles.
16
20
Calculations have been performed to estimate the time needed for these [4Fe4S] repair
proteins to search for DNA damage, and the results indicate that 3D random collisions, 2D
sliding and hopping, and facilitated diffusion are not fast enough to explain the quick action
of these proteins.
21
Given the low copy number of unique DNA-processing [4Fe4S]
enzymes, ranging from 500 to as low as 10 per cell,
22
,
23
and the vast number of DNA
lesions, we have proposed that these proteins aid one another through signaling using DNA-
mediated charge transport (CT) between their [4Fe4S] clusters to search for and locate DNA
damage sites more efficiently.
21
,
24
,
25
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The highly ordered
π
-stacking structure of aromatic bases enables efficient CT through
double-stranded DNA (dsDNA).
26
28
One single base pair mismatch, lesion, or abasic site
that disrupts
π
-stacking can attenuate DNA-mediated CT significantly.
29
31
Ground state
CT mediated by dsDNA has been measured over 100 base pairs of length, and, given a very
shallow distance dependence, there is no clear indication for the maximum distance that
charge can move through dsDNA.
32
Experiments with DNA repair proteins in the native 2+ oxidation state for the cluster show
that CT-active proteins containing [4Fe4S] clusters localize in the vicinity of damage.
24
,
25
,
33
The DNA-mediated redox properties of [4Fe4S] proteins have been established
in vitro
using biochemical assays and DNA-modified electrodes. These experiments show that the
[4Fe4S] cluster within a protein can be reduced to the 2+ state or oxidized to the 3+ state by
CT through a DNA duplex. The efficiency of this process is diminished when the DNA
contains a mismatch or abasic site located between the protein and the electrode.
29
31
Furthermore these experiments have found that binding to DNA shifts the [4Fe4S]
2+/3+
couple negative by about 200 mV to ca. +80 mV vs. NHE.
34
,
35
This shift
thermodynamically suggests a corresponding shift in the DNA-binding affinity of proteins
with an oxidized [4Fe4S] cluster by at least two orders of magnitude when compared to the
reduced protein. However, no direct measurement of the DNA-binding affinities of [4Fe4S]
repair proteins with intact clusters in both the reduced and oxidized state have been
obtained. Also, it is unclear how much of this potential shift is caused by the change in
affinity, as it may be influenced by a variety of other factors, such as a reorganization of
residues in the local environment of the cluster. Thus, it is essential to measure directly the
change in affinity associated with [4Fe4S] redox state.
In this report, we systematically vary the oxidation state of the [4Fe4S] cluster and measure
how the redox state of the metallocofactor modulates DNA binding and, in turn, controls
DNA damage detection activity of repair proteins. We specifically interrogate how oxidative
stress influences the ability of [4Fe4S]-containing DNA repair proteins, Endonuclease III
(EndoIII) and DinG, to bind preferentially to a 3 kilobase pairs (kbp) DNA duplex with a
single-base mismatch
in vitro.
EndoIII is a base excision repair (BER) glycosylase that
repairs oxidized pyrimidines in
Escherichia coli
(
E. coli
).
9
DinG is a DNA helicase that
unwinds R-loops caused by invasion of DNA duplexes by nascent mRNA strands at
transcription bubbles in
E. coli
.
36
Both proteins have been identified to contain high-
potential [4Fe4S] clusters (HiPIPs), which cycle between the [4Fe4S]
3+
and [4Fe4S]
2+
states
at physiological potentials when bound to dsDNA. Previous reports have demonstrated that
EndoIII and DinG can interact with each other in a synergistic fashion by localizing on
mismatch-containing DNA strands
in vitro
and rescuing
E. coli
growth
in vivo
.
33
,
37
Neither
protein binds preferentially to a mismatch site, so the preference for binding the mismatch-
containing strand is attributed instead to the ability of the mismatch to inhibit DNA CT.
21
,
25
,
38
,
39
Here, we selectively oxidize and reduce the [4Fe4S] metallocofactors in EndoIII
and DinG using electrochemical methods instead of adding chemical reagents, which could
damage the proteins and DNA strands. We then utilize atomic force microscopy (AFM) to
examine the DNA-protein interactions with varying amounts of [4Fe4S] cluster oxidation.
We further develop an electrostatic model based upon the electrostatic interactions between
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DNA and [4Fe4S] cluster proteins to understand the change in protein binding with
oxidation state.
EXPERIMENTAL SECTION
General Procedures
Chemicals were obtained from commercial sources (Sigma Aldrich, Fisher Scientific, VWR,
and New England Biolabs) and used without further purification unless otherwise specified.
Protein buffer (pH 7.5, 20 mM NaH
2
PO
4
, 100 mM NaCl, 5% glycerol, 1 mM EDTA) and
DNA buffer (pH 7.0, 5 mM NaH
2
PO
4
, 50 mM NaCl) were prepared using Milli-Q water
(>18 M
Ω
cm). Solutions were degassed and sparged with Ar overnight prior to conducting
electrochemical experiments and preparing AFM samples inside an anaerobic chamber (Coy
Lab Products). Experiments performed were replicated at least three times using different
samples, and data presented are from representative trials.
Preparation of DNA-modified Electrodes for Electrochemical Studies
DNA sequences were prepared using phosphoramidites (Glen Research) on a DNA
Synthesizer (Applied Biosystems 3400) or purchased from Integrated DNA Technologies
and then purified by high performance liquid chromatorgraphy (HPLC, HP 1100, Agilent).
Thiolated DNA was prepared according to established methods by modifying the 5
end
using a C6 S–S phosphoramidite.
32
Deprotection, purification, quantification, matrix-
assisted laser desorption ionization (MALDI) characterization using a Autoflex MALDI
TOF/TOF (Bruker), annealing of DNA, fabrication of Au electrodes, and preparation of the
DNA-modified Au electrodes were performed as described previously.
32
,
40
,
41
The Au
electrodes were assembled and then incubated in a solution (200 μL) of thiol-modified
dsDNA (50 μM) in DNA buffer for 24 h at room temperature in the dark. The electrode
modified with a monolayer of thiolated-DNA was then backfilled with mercaptohexanol (1
mM, 200 μL) for 45 min, washed three times with DNA buffer or protein buffer prior to
adding protein samples to the DNA-modified Au surfaces.
DNA sequences used for EndoIII electrochemistry:
35
HS-C6-5
-GT GAG CTA ACG TGT
CAG TAC-3
3
-CA CTC GAT TGC ACA GTC ATG-5
DNA sequences used for DinG electrochemistry:
33
HS-C6-5
-GT GCT GCA ACG TGT
CTG CGC-3
3
-CA CGA CGT TGC ACA GAC GCG AGA GCA GAC GTC AGA-5
(HS-C6 = hexanethiol linker)
Protein Preparation and Characterization
Wild-type proteins (EndoIII and DinG) were prepared as described previously.
33
Crude
proteins were harvested from cells and purified using fast protein liquid chromatography
(FPLC, Bio-Rad) at 4 °C. Protein concentration was quantified based on the [4Fe4S] cluster
absorbance (
ε
410
= 17000 M
−1
cm
−1
)
42
using a 100 Bio UV-visible spectrophotometer
(Cary, Agilent). Cluster loading was over 70%. Protein samples (5 μM) were sealed in
Teflon-capped cuvettes under anaerobic condition and characterized at room temperature on
a Model 430 circular dichroism spectrometer (Aviv). Continuous-wave electron
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paramagnetic resonance (EPR) and pulse electron spin echo envelope modulation (ESEEM)
experiments were conducted on an EMX X-band CW-EPR Spectrometer (Bruker) and an
ELEXSYS-E580 X,Q-band Pulse EPR Spectrometer (Bruker) at 10 K with samples sealed
in EPR tubes in the glove bag.
Electrochemistry
Electrochemical studies were performed as described previously.
34
,
41
,
42
Electrochemical
experiments were carried out in a three-electrode set-up under an anaerobic atmosphere. The
working electrode was a DNA-modified Au electrode, the counter electrode was a piece of
freshly-polished Pt wire separated from the solution by an agarose gel tip filled with NaCl (3
M), and the reference electrode was a Ag/AgCl electrode stored in saturated NaCl solution
modified with an agarose gel tip filled with NaCl (3 M). All redox potentials were converted
to and reported versus the NHE scale.
Cyclic voltammetry, square wave voltammetry, and chronoamperometry were carried out
using a 760 D Electrochemical Workstation (CH Instruments) at room temperature inside an
anaerobic chamber. The atmosphere of the anaerobic chamber (< 1 ppm O
2
, ca. 3.4% H
2
)
was monitored using a CAM-12 O
2
and H
2
sensor (Coy Lab Products). The chamber was
maintained O
2
-free by using two ventilated Pd catalyst packs (Coy Lab Products). Protein
sample was placed on an electrode surface and successive potential cycling treatments
between +0.4 V and −0.2 V vs. NHE at a scan rate of 100 mV/s using cyclic voltammetry
were taken until the signal stabilizes. Bulk electrolytic oxidation and reduction of proteins
were conducted by holding the electrode potential at +250 mV and −50 mV respectively vs.
NHE for 25 min which is when the current stabilizes at a value similar to the background
current obtained in a trial without protein in solution. The amount of charge passed during
bulk electrolysis was cal3 culated by integrating the area under the background-subtracted
curve in the current vs. time chronoamperometric plot. The integrated charge was then
divided by the Faraday’s constant (96485 C/mol) to convert to the total number of moles of
electrons passed.
43
Since a [4Fe4S] cluster undergoes a 1-electron redox process,
44
the
number of moles of electrons passed equals to the number of proteins oxidized or reduced.
The number of moles of proteins present in the solution prior to bulk electrolysis was
calculated from the molarity and volume of the protein solution used. The bulk electrolysis
yield was then computed as the percentage of [4Fe4S] cluster protein oxidized or reduced in
the whole protein sample.
Synthesis of DNA Strands for AFM Experiments
Short well-matched DNA duplex, long well-matched DNA duplex, and long DNA duplex
with a C:A mismatch engineered in the middle were prepared using published protocols.
21
,
25
,
33
Briefly, four primers were synthesized, phosphorylated, and purified using phenol–
chloroform extraction followed by ethanol precipitation. After drying under vacuum
overnight, primers were used in PCR reactions using pUC19 as a template to generate two
DNA duplexes containing 14-nucleotide single-strand overhang. Short and long duplexes
were annealed and ligated together. Incomplete reaction resulted in a mixture of ligated
(3767 bp, ca. 1.2 μm in length) and unligated (1610 bp and 2157 bp, ca. 0.5 μm and 0.7 μm
in length) dsDNA samples that were identical with the exception of the presence of the
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mismatch site engineered at the overhang region. The single-strand overhang of unligated
dsDNA was conjugated to complementary DNA strands prior to AFM experiments.
AFM Experiments
AFM was conducted following protocol reported previously.
21
,
25
,
33
Briefly, mica surfaces
were freshly cleaved with tape. Protein stock solution containing either 5 μM EndoIII or 2
μM DinG was collected after bulk electrolysis. Stock DNA solution (6 ng/μL) contained the
mixture of ligated duplexes and the two unligated duplexes in Tris elution buffer (EB,
Qiagen). A solution with a final protein concentration (12 nM) and a final DNA
concentration (1 ng/μL) was prepared and incubated at room temperature for 1 h to allow for
the loading of protein onto DNA to reach equilibrium under an anaerobic atmosphere and
minimize cluster degradation. MgCl
2
(200 mM, 1.5 μL per 25 μL total volume) was added to
promote DNA adsorption on mica for AFM experiments. After pipetting 10 μL of DNA/
protein/MgCl
2
solution onto a mica surface and incubating for 2 min, a continuous stream of
deoxygenated Milli-Q water (2 mL) was slowly poured over the top portion of the modified
mica surface while holding the piece of mica in a vertical position to linearize the DNA. A
piece of kimwipe was used to dab dry the bottom edge of the mica surface. The surface was
dried using a stream of N
2
flowing in the same direction as the water rinse for 2 min. pUC19
(2,686 bp, ca. 0.9 μm) linearized by HindIII (New England Biolabs) was used instead of the
ligated and unligated DNA to quantify how the DNA-binding activity of EndoIII changes as
a function of the ratio of oxidized [4Fe4S]
3+
cluster in the protein sample.
AFM Instrumentation
FESPA-V2 AFM tapping mode probes (Bruker Nano, Inc.) with a mean force constant of
2.8 N/m and mean resonance frequency of 75 kHz were used in a MFP-3D AFM (Asylum
Research). Images were captured in air with scan areas of 3×3 μm
2
in tapping mode at a
scan rate of 1 Hz in order to obtain images of quality high enough for AFM redistribution
assay analysis (512 pixels/line, 512 lines/image). The images were collected and analyzed
blind.
Differential Binding Density Ratio Calculations
WSxM software (Igor Pro) was used to measure DNA contour lengths and height profiles of
the proteins as described previously.
21
,
25
,
33
DNA and proteins were identified using the
relative differential height profiles between protein and DNA. For each data set, images from
at least three independent samples were analyzed, compared, and pooled (> 400 long and
short duplexes). Distinguishable DNA and proteins were counted by hand. Duplexes that
were overlapped, indistinguishable, or cut off by the edge of the image were excluded from
our counting procedure. For each sample, the binding density ratio,
r
, (see Eq. 1) is defined
as the ratio of the proteins bound on long duplexes divided by proteins bound on short
duplexes, which serve as an internal normalization factor to correct changes in concentration
across samples that may confound results. Data presented are from representative trials, and
error bars represent standard error of all trials based on the total number of proteins observed
(n > 200 for all experiments). The differential binding density ratio,
dr
, (see Eq. 2) is
calculated by dividing the
r
from the mismatched sample by the
r
from the well-matched
sample.
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r =
#
o f proteins on long duplexes
#
o f proteins on short duplexes
Eq. 1
d
r
=
r
mismatched
r
well
matched
=
#
o f proteins on mismatched long duplexes
#
o f proteins on short duplexes
#
o f proteins on well
matched long duplexes
#
o f proteins on short duplexes
Eq. 2
Microscale Thermophoresis
Microscale thermophoresis (MST) was carried out using a Monolith NT.115 series
instrument (NanoTemper) at 21 °C. Oligonucleotides (21 bp in length) based on the DNA
sequence used in the AFM studies were synthesized and annealed to generate well-matched
DNA of 21 bp in length. For measurements of EndoIII primarily in the reduced state, native
EndoIII with His
6
-tag (200 nM, 100 μL) was mixed with MO-L008 His-tag labeling kit
RED-tris-NTA (100 nM, 100 μL, NanoTemper) for 30 min at room temperature. The sample
was centrifuged at 4 °C and 15,000 g for 10 min. Serial dilution of dsDNA as titrants was
performed in PCR 8-strip tubes with individual caps (USA Scientific) following the user
manual provided by NanoTemper. Labeled protein (50 nM) was mixed with DNA titrant in
the dark for 10 min. Samples with a final dye concentration of 25 nM were then loaded into
MO-K003 Monolith NT.115 hydrophobic capillaries (NanoTemper) and were measured at
60% LED and 40% MST power. DNA buffer with 0.05% Tween 20 was used for MST
studies. For measurements of EndoIII primarily in the oxidized state, EndoIII (1 μM, 200
μL) was oxidized in the absence of O
2
using DNA-modified electrode previously described.
Oxidized EndoIII was labeled with RED-tris-NTA under anaerobic condition in an
aluminum-wrapped Eppendorf tube. MST samples were loaded into hydrophobic capillary
tubes inside a glove bag in the dark. Capillaries were sealed with HR4-328 capillary wax
(Hampton Research) using a hand-held wax pen (Hampton Research) in the absence of O
2
and light.
DNA sequences used for MST measurements: 5
-ACT GAA CTC TGT ACC TGG CAC-3
3
-TGA CTT GAG ACA TGG ACC GTG-5
Electrostatic Modeling
Distances between the [4Fe4S] cluster of EndoIII and the phosphate groups on the backbone
of the DNA-bound EndoIII in the native state were obtained from a published crystal
structure (PDB: 1ORN).
45
The 1ORN crystal structure also provided the number of
phosphates interacting with protein residues. The dielectric constant of the portion of
EndoIII of interest was estimated by calculating the average dielectric constant of the amino
acid residues
46
surrounding the [4Fe4S] cluster and in between the [4Fe4S] cluster and the
bound DNA strand. In this electrostatic model, the [4Fe4S] cluster and the phosphate groups
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on the DNA backbone were modeled as point charges. The potential energy (PE) between
two charged particles was calculated using Eq. 3
PE
=
(
q
1
)(
q
2
)
4
π ε
0
ε
r
d
Eq. 3
with
q
= charge,
ε
0
= permittivity of free space,
ε
r
= dielectric constant, and
d
= distance
between charges. The difference in PE between EndoIII in the reduced and oxidized states
was computed by calculating the difference between the total PE for the [4Fe4S]
3+
case and
the total PE for the [4Fe4S]
2+
case. The experimental energy difference between the 3+ case
and the 2+ case was calculated using the Nernst equation by converting the redox potential
shift recorded using electrochemical techniques.
34
The electrostatic model was supported by
comparing the modeled PE and the experimental energy difference.
RESULTS
Oxidation and Reduction of [4Fe4S] Proteins using Electrochemical Methods
Cyclic voltammetry (CV) was used to investigate the electrochemical properties of EndoIII
on DNA-modified Au electrodes (Figure 1). These data, along with square wave
voltammetry and differential pulse voltammetry measurements (Figures S1–2) show that
within our potential window EndoIII exhibits a single redox couple with a DNA-bound
midpoint potential of about +80 mV vs NHE, consistent with previous measurements of the
[4Fe4S]
2+/3+
redox couple in EndoIII when associated to DNA.
34
,
35
,
39
Solutions of primarily reduced or primarily oxidized EndoIII with an intact [4Fe4S] cluster
were generated using bulk electrolysis and confirmed using electron paramagnetic resonance
(EPR) and circular dichroism (CD). Randles-Sevcik analysis (Figures S3–4) demonstrates
that there is exchange between proteins in solution and on the surface of the DNA-modified
electrode so that a constant applied potential could oxidize or reduce the majority of proteins
in the sample. The CVs shown in Figure 1 identify potentials at which the [4Fe4S] clusters
of the proteins can be oxidized or reduced, which allow us to conduct bulk electrochemical
oxidation and reduction of EndoIII at +250 mV and −50 mV vs. NHE, respectively. Similar
electrolysis experiments were utilized to examine primase using its [4Fe4S] cluster as a
redox switch.
47
Figure 1 also shows the oxidation of EndoIII on a DNA-modified electrode
held at a constant potential for 25 min to drive electrolysis to completion. Applying an
oxidizing or reducing potential to a solution with protein present results in a larger
magnitude current than measured in the buffer control (Figure S5). This difference is
indicative of the proteins being oxidized or reduced on the DNA-modified electrode surface,
and the current decreases over time as the oxidation or reduction approaches completion (>
99% yield). We then utilized EPR to characterize the reduced and oxidized EndoIII
generated by bulk electrolysis (Figure S6). The reduced EndoIII is EPR-silent and the
oxidized EndoIII exhibits an EPR signal with a g value of 2.07, which are consistent with
intact [4Fe4S]
2+
and [4Fe4S]
3+
clusters, respectively.
48
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Binding Affinity Measurements Comparing EndoIII with Oxidized and Reduced [4Fe4S]
Cluster
AFM was used to explore how the DNA-binding activity of EndoIII changes as the amount
of oxidized and reduced clusters in the protein sample was varied (Figure 2). We then
recorded the number of EndoIII present on the linearized pUC19 plasmid DNA as a function
of the proportion of oxidized [4Fe4S]
3+
cluster in the protein sample (Table S1). As the ratio
of EndoIII with oxidized [4Fe4S]
3+
metallocofactors increases, the number of EndoIII
bound on DNA increases (Figure 2). This trend indicates that oxidized EndoIII binds more
tightly to DNA than does the reduced protein. We then utilized electrophoretic mobility shift
assay (EMSA), isothermal titration calorimetry (ITC), and microscale thermophoresis
(MST) to probe the non-specific DNA-binding affinity of EndoIII to well-matched dsDNA
of 21 bp in length. In all three cases, we observed an increase in binding affinity of EndoIII
to dsDNA upon oxidizing the [4Fe4S] metallocofactor from the 2+ state to the 3+ state
(Table 1). However, ITC is not anaerobic and EMSA generates O
2
in situ
, both of which
result in cluster degradation that prevent accurate binding affinity measurements (see S.I. for
further discussion). To prevent cluster degradation, we utilized MST for anaerobic binding
affinity measurement.
49
MST results show that EndoIII predominantly in the reduced state
exhibits a K
D
of 6.1 μM ± 0.9 μM for unmodified duplex DNA and EndoIII predominantly
in the oxidized state displays a K
D
of 0.011 μM ± 0.002 μM (Figures 3 and S7). Thus, we
observed a 550 ± 130 times increase in DNA-binding affinity of EndoIII upon oxidation,
which further supports the trend observed using AFM that the EndoIII occupancy on DNA
increases upon oxidation.
Electrostatic DNA-Protein Binding Model
To understand the origin of the redox-modulated affinity, we developed an electrostatic
model that investigates the perturbation in the electrostatic interactions between DNA and
proteins upon the addition or removal of an electron at the [4Fe4S] metallocofactor (Figure
S8). This electrostatic model incorporates atomic distances obtained from crystallographic
data and knowledge regarding the DNA-binding pocket from published literature.
9
,
45
,
50
The
crystal structure (PDB: 1ORN) revealed that eight negatively-charged phosphates on the
DNA backbone not bound by cations interact with the amino acid residues of EndoIII.
Another crystallographic study demonstrated that a high-potential iron-sulfur protein
(HiPIP) undergoes only minor structural changes upon toggling the redox state of the
[4Fe4S] cluster between 2+ and 3+.
51
Therefore, we assumed the coordinates of the atoms in
the EndoIII protein structure are the same for both the [4Fe4S]
3+
and [4Fe4S]
2+
cases. The
binding energy resulting from electrostatic interactions between the positively charged
[4Fe4S] cluster and the negatively-charged phosphate groups on the DNA backbone are
calculated to be ca. 7 kcal/mol, comparable to the energy change measured from the redox
potential shift of EndoIII upon binding DNA using the Nernst equation, ca. 5 kcal/mol.
52
The model ΔPE for MutY also recapitulates the energy difference between the 2+ and 3+
states computed from the redox potential shift obtained using electrochemical techniques.
35
This result emphasizes that our electrostatic model is general and is readily applicable to
other DNA-processing enzymes carrying [4Fe4S] metallocofactors. This model shows that
the difference in binding affinity for the two oxidation states, [4Fe4S]
2+
and [4Fe4S]
3+
, can
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be explained primarily by the electrostatic interactions between the cluster and DNA without
a substantial protein structural change.
Signaling Between Oxidized and Reduced EndoIII to Search for DNA Damage
We then examined the effect of the redox state of the [4Fe4S] metallocofactor on the ability
of EndoIII to differentiate between well-matched (WM) and mismatched (MM) DNA
strands by AFM. Solutions of EndoIII with controlled amounts of oxidized [4Fe4S]
3+
cluster and reduced [4Fe4S]
2+
cluster were incubated with two different mixtures of DNA
and then the two sets of AFM results were compared. The first DNA mixture contained long
and short duplexes with no mismatches, while the second DNA mixture contained both short
DNA duplexes with no mismatches and long DNA duplexes with a single C:A mismatch,
which is not a native substrate for EndoIII but does inhibit DNA CT, engineered in the
middle of the strand. The short WM DNA duplexes are identical in the two sets of
experiments so the binding distribution profiles across the two data sets can be normalized
and compared to one another (Tables S2–3). Figure 4 shows a bar graph summarizing the
ability for solutions of EndoIII with varying proportions of oxidized proteins to bind
preferentially to DNA with a single-base mismatch. Here we observed that the ability for
EndoIII to differentiate between MM and WM DNA strands decreases as the percentage of
oxidized protein in the sample increases. It should be noted that these data could also be
used to determine the change in binding affinity with oxidation. On this basis, we found a 4–
30 fold increase in DNA-binding affinity upon oxidizing the [4Fe4S] cluster of EndoIII. The
trend observed is consistent with that obtained using other techniques, but the magnitude of
change is significantly smaller. This difference likely can be attributed to the protein-DNA
sample not being at equilibrium in the AFM experiment, because of kinetic limitations
associated with tight DNA binding, precluding rapid, full redistribution of proteins along the
DNA.
Signaling Between EndoIII and DinG to Search for DNA Damage
This change in binding density ratio, associated with a change in oxidation state, could be
used as a diagnostic of oxidation state in the AFM experiment and therefore a means to
identify redox signaling between proteins. We evaluated whether DinG, another DNA-
processing enzyme containing a [4Fe4S] cluster, exhibits the same redox-modulated ability
as EndoIII to bind preferentially to DNA duplexes with an intervening lesion. We chose to
incorporate DinG because it has been shown to carry out DNA signaling with EndoIII; both
proteins in the native oxidation state search for DNA damage
in vitro
and there is evidence
that EndoIII helps DinG rescue defective growth
in vivo
.
33
DinG exhibits a DNA-bound
redox potential comparable to that of EndoIII (Figures S9–12). We subjected DinG to bulk
electrolysis conditions similar to EndoIII to generate DinG with intact oxidized and reduced
[4Fe4S] metallocofactors (Figure S13). Analogous to EndoIII, oxidizing DinG decreases its
ability to bind preferentially to DNA duplexes with a single-base mismatch (Figure 5 and
Tables S4–5).
Mixtures of reduced DinG and oxidized EndoIII, as well as mixtures of oxidized DinG and
reduced EndoIII, were then prepared to address whether a reduced protein can signal with a
different oxidized protein (Tables S6–9). This cross experiment includes four scenarios: two
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contain proteins at the 33% overall oxidized state, and two contain proteins at the 66%
overall oxidized state. Figure 5 shows the results of the cross experiments. When the overall
protein oxidation is either 33% and 66%, EndoIII and DinG exhibit intermediate binding
density ratios in between the values obtained for predominantly reduced and predominantly
oxidized protein samples. These findings demonstrate that reduced proteins from different
repair pathways can undergo DNA-mediated redox signaling with oxidized proteins to
collaboratively search for and preferentially bind to DNA duplexes with a single-base
mismatch.
DISCUSSION
Redox-Active [4Fe4S] Cluster of Repair Proteins Functions as a DNA-Binding Affinity
Switch
Non-specific binding to DNA is an important first step in the damage search and repair
process.
3
5
,
24
The binding affinity measurements reported in this work clearly indicate that
the oxidized protein has an increased binding affinity for DNA relative to the reduced
protein. This direct measurement of an increased affinity is consistent with electrochemical
results that previously demonstrated binding to DNA shifts the redox potential of the
[4Fe4S]
2+/3+
couple of the cluster negative by about 200 mV,
35
,
53
,
54
which indirectly
showed that oxidation increases the protein/DNA binding affinity by at least two orders of
magnitude.
34
We devised an electrostatic model to understand the underlying chemistry that dictates the
apparent change in DNA-binding affinity. We assumed that changing redox state of the
[4Fe4S] cluster does not significantly alter the structure of the protein, consistent with
minimal structural changes observed in recent crystallographic studies on a HiPIP that is
reduced or oxidized,
51
EndoIII with and without DNA,
9
,
45
,
50
and MutY spectroscopically
examined with and without a cluster.
15
Utilizing these structural data, we developed an
electrostatic model to understand how the redox state of the [4Fe4S] cluster affects the
charge-charge interaction. Crystallographic data confirms that EndoIII binds to the
negatively-charged phosphate groups on the DNA backbone via a series of positively-
charged residues.
45
Our electrostatic model is general and is readily applicable to other
DNA-processing enzymes carrying [4Fe4S] metallocofactors. Our model demonstrates that
the redox-modulated DNA-binding affinity can be fully explained by the electrostatic
interactions between the [4Fe4S] cluster and the phosphate groups on the DNA backbone
without a significant structural change between oxidized and reduced DNA-bound forms.
The lack of a necessary conformational change may be critical in the detection and location
of lesions.
Redox-Modulated DNA-Binding Affinity of [4Fe4S] Repair Proteins Regulates DNA-
mediated CT Signaling
AFM experiments reveal that the oxidation state of the [4Fe4S] cluster influences the ability
of the protein to localize on DNA duplexes with single-base mismatches. The total amount
of oxidation is important for the efficiency of this DNA damage sensing process. Despite
starting with oxidized protein that is unable to localize preferentially on mismatch-
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containing DNA, the addition of reduced protein with different enzymatic function is able to
achieve preferential binding. Intriguingly, the amount of oxidized [4Fe4S] cluster among all
proteins in a mixture is a better predictor of damage recognition than the identity of the
proteins with initially reduced or oxidized [4Fe4S] metallocofactors; thus it can be used as a
diagnostic of signaling between proteins.
To mimic cellular oxidative stress, our group previously used H
2
O
2
to oxidize the [4Fe4S]
cluster in EndoIII. These experiments found that H
2
O
2
increases the preference of [4Fe4S]
cluster proteins for DNA duplexes containing a mismatch.
21
These results are fully
consistent with the idea that repair proteins with [4Fe4S]
2+
clusters sense and respond to
oxidative stress via cluster oxidation to the 3+ state. However, it is unclear what other effects
the H
2
O
2
may have on the proteins and DNA in this system.
5
,
24
The use of chemical
oxidants such as K
3
Fe(CN)
6
and H
2
O
2
degrade [4Fe4S] clusters.
6
,
42
Also, the oxidation
extent of the [4Fe4S] cluster in the protein sample cannot be accurately quantified by using
an excess amount of H
2
O
2
as the oxidant. The electrochemical oxidation and reduction
presented here provides a milder and well-controlled condition that allows for changing the
redox state of [4Fe4s] clusters. Together, these data confirm that oxidative stress can
generate repair proteins with [4Fe4S]
3+
clusters that can aid the DNA damage search. It is
important to note that the short incubation time in both of these experiments, which was
required for sample stability, means that the protein-DNA binding may not be at
equilibrium. If anything, giving more time to equilibrate should result in higher preferential
binding to the duplex with the mismatch.
These observations using AFM are consistent with a DNA damage search scheme based on
DNA-mediated redox signaling between proteins with oxidized and reduced [4Fe4S]
clusters (Figure 5). DNA-mediated CT can occur between two proteins and, our data shows
conclusively, this CT results in a change in affinity for each of the proteins. Incorporating a
single C:A mismatch within a DNA duplex, even though it is not a preferred substrate for
either EndoIII or DinG,
27
,
31
is able to increase the number of proteins bound to that duplex
relative to a duplex with no mismatches. In these experiments the mismatch only serves to
disrupt DNA CT between proteins and, therefore, DNA CT prevents the exchange of affinity
that results from changing the redox state of the [4Fe4S] clusters. This change in redox state
increases the effective affinity of these proteins for a duplex containing a mismatch because
it allows for oxidized proteins to stay in the vicinity of the mismatch, or potentially any
lesion that disrupts DNA CT, without being reduced and released from the duplex by another
protein. These data bolster the claim that proteins, even with completely different cellular
roles, can work in concert to aid their search for DNA damage by reducing and oxidizing
one another via long-range CT through DNA.
55
57
Redox-Signaling Damage Search Model
DNA-mediated CT significantly expedites the search for DNA damage. Charge can be
transported through large regions of DNA much faster than proteins can translocate,
especially when the nuclear medium and DNA itself are crowded by other proteins.
21
If a
high-affinity, oxidized protein is bound to a region with no lesion, the protein may quickly
become reduced by another protein along the duplex, and the decreased affinity will allow
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for it to release DNA and scan elsewhere (Figure 5). If, however, there is a lesion disrupting
DNA CT, the protein will remain oxidized and associated to DNA. They can utilize both
DNA CT and translocation making the search process faster and more efficient.
DNA-Mediated Redox Signaling Enables Low Copy Number Proteins to Efficiently Find
Their Substrate
A growing number of proteins that process DNA have been discovered to contain redox-
active [4Fe4S] clusters.
7
10
,
20
Our data show that the oxidation state of the [4Fe4S] clusters
is integral to the ability of proteins to search for DNA damage, but that the identity of the
protein is unimportant for DNA-CT. The DNA-binding affinity depends on the oxidation
state of the [4Fe4S] cluster in both EndoIII and DinG and likely holds true for other [4Fe4S]
proteins. Intriguingly, this model suggests that DNA-processing proteins with different
enzymatic functions may collaborate with each other to find damage so long as the [4Fe4S]
clusters are in different redox states. Because the redox-modulated affinity relies purely on a
change in electrostatic interactions and not on protein-specific changes in structure, the
DNA-mediated redox signaling strategy between proteins containing [4Fe4S] clusters likely
benefits the efficien9 cy of all DNA-processing proteins containing [4Fe4S] to find and
repair damage.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We gratefully recognize the NIH (GM61077) for financial support. E.C.M.T. appreciates the Croucher Foundation
for a post-doctoral fellowship. T.J.Z. is an NSF fellow (DGE-1144469). E.C.M.T. also thanks Zhou for assisting in
the protein purification process and Dr. Deng for providing Au working electrodes. We are also grateful to the
Caltech Center for the Chemistry of Cellular Signaling for instrumentation.
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Figure 1.
Electrochemistry of EndoIII on DNA-modified electrodes. (a) CVs at a scan rate of 100
mV/s and (b) bulk electrolysis held at +250 mV vs. NHE in DNA buffer (pH 7.0, 5 mM
NaH
2
PO
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, 50 mM NaCl) without (black) and with 1 μM EndoIII (red) using DNAmodified
electrodes passivated with mercaptohexanol.
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Figure 2.
AFM visualization of EndoIII binding to DNA. (a) Visualization of proteins on DNA
duplexes using AFM. The relative height of proteins allows for them to be distinguished
from DNA and counted. (b) Plot of the amount of EndoIII bound on a DNA strand as a
function of the extent of oxidation of the protein samples. The 33% and 66% oxidized
EndoIII samples were prepared by mixing reduced and oxidized EndoIII in a 2:1 and 1:2
ratios, respectively. Curved lines in the schematic on the right represent linearized pUC19
DNA duplexes.
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Figure 3.
Microscale thermophoresis (MST) results of EndoIII. MST plots of reduced (green) and
oxidized (red) EndoIII binding to DNA. A 21-mer duplex was used as the titrant. A 550-fold
increase in binding affinity was observed for EndoIII upon oxidation.
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Figure 4.
AFM redistribution assay of EndoIII samples oxidized to various extent. The 33% and 66%
oxidized EndoIII samples were prepared by mixing reduced and oxidized EndoIII in a 2:1
and 1:2 ratios, respectively. In the schematic on the right, short curved lines, long curved
lines, and long curved lines with a red feature in the middle represent short well-matched
DNA duplexes, long well-matched DNA duplexes, and long DNA duplexes with a C:A
mismatch engineered in the middle.
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Figure 5.
AFM redistribution assays for (a) oxidized and reduced DinG, (b) oxidized EndoIII with
reduced DinG, and (c) oxidized DinG with reduced EndoIII. The 33% and 66% oxidized
samples in (b) were prepared by mixing reduced DinG and oxidized EndoIII in a 2:1 and 1:2
ratios, respectively. The 33% and 66% oxidized samples in (c) were prepared by mixing
reduced EndoIII and oxidized DinG in a 2:1 and 1:2 ratios, respectively. (d) Our proposed
DNA damage search model for DNA-processing enzymes carrying redox-active [4Fe4S]
clusters that uses DNA-mediated CT as the first step of damage detection. Reduction of the
[4Fe4S] cluster through unperturbed WM DNA allows for proteins to release from the DNA
and search elsewhere for damage. If, however, damage is present along the DNA, the protein
will not be reduced and will localize in the vicinity of the DNA damage.
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