DNA-Mediated Signaling by Proteins with 4Fe
−
4S Clusters Is
Necessary for Genomic Integrity
Michael A. Grodick, Helen M. Segal, Theodore J. Zwang, and Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
*
S
Supporting Information
ABSTRACT:
Iron
−
sulfur clusters have increasingly been
found to be associated with enzymes involved in DNA
processing. Here we describe a role for these redox clusters in
DNA-mediated charge-transport signaling in
E. coli
between
DNA repair proteins from distinct pathways. DNA-modi
fi
ed
electrochemistry shows that the 4Fe
−
4S cluster of DNA-
bound DinG, an ATP-dependent helicase that repairs R-loops,
is redox-active at cellular potentials and ATP hydrolysis
increases DNA-mediated redox signaling. Atomic force
microscopy experiments demonstrate that DinG and Endo-
nuclease III (EndoIII), a base excision repair enzyme, cooperate at long-range using DNA charge transport to redistribute to
regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs
within the cell and, remarkably, is required for cellular viability under conditions of stress. Silencing the gene encoding EndoIII in
a strain of
E. coli
where repair by DinG is essential results in a signi
fi
cant growth defect that is rescued by complementation with
EndoIII but not with an EndoIII mutant that is enzymatically active but unable to carry out DNA charge transport. This work
thus elucidates a fundamental mechanism to coordinate the activities of DNA repair enzymes across the genome.
■
INTRODUCTION
Iron
−
sulfur clusters are increasingly being found in proteins
that are tasked with maintaining the
fi
delity of the genome.
1
−
3
These clusters were
fi
rst observed in DNA-binding proteins in
the base excision repair (BER) glycosylase, Endonuclease III
(EndoIII).
4
More recently, 4Fe
−
4S clusters have been found in
a range of DNA repair and DNA processing enzymes
including helicases, DNA and RNA polymerases, DNA
helicase-nucleases, and DNA primases from across the
phylogeny.
2,4
−
13
Many of the enzymes that have been shown
to contain these clusters are genetically linked to human
diseases, such as early onset breast cancer and Fanconi
’
s
anemia, yet the proteins perform immensely di
ff
erent functions.
The clusters do not participate in catalysis in these proteins,
2,3,5
though DNA binding may be a
ff
ected by perturbing the
cluster.
14
Recently, studies focusing on the biogenesis of iron
−
sulfur clusters have revealed that disruption of iron
−
sulfur
cluster assembly proteins in eukaryotic cells leads to nuclear
genomic instability and defects in DNA metabolism,
replication, and repair.
15
−
17
The ubiquity of these complex
cofactors suggests an essential and shared role for their
presence in DNA processing enzymes.
We have considered that the 4Fe
−
4S clusters in DNA repair
enzymes may serve as redox cofactors, much as 4Fe
−
4S clusters
do in other enzymes within the cell.
1,18
Most of our work has
focused on
E. coli
EndoIII, where the 4Fe
−
4S cluster was
fi
rst
found. Although a redox role for the cluster was considered,
4
the 4Fe
−
4S cluster in EndoIII is redox-inactive at typical
cellular potentials. We showed, however, that DNA binding
shifts the redox potential of the cluster to 80 mV vs the normal
hydrogen electrode (NHE), moving the 3+/2+ redox couple
into the physiological regime.
19
Strikingly, we have now seen
that 4Fe
−
4S clusters in other repair proteins share this DNA-
bound potential of
∼
80 mV versus NHE.
20,21
We have
proposed that these clusters are utilized for DNA-mediated
charge-transport (CT) chemistry as a
fi
rst step in the search for
DNA lesions to repair.
18,22
Indeed we have explored how
EndoIII and another BER glycosylase with a 4Fe
−
4S cluster,
MutY, may use DNA CT cooperatively as a
fi
rst step in
repair.
22
Here we explore how DNA CT may be utilized more
generally in
E. coli
for interprotein signaling between repair
pathways to maintain the integrity of the genome.
The chemistry of DNA CT o
ff
ers a powerful tool to probe
the integrity of duplex DNA. It has now been well documented
that DNA can conduct charge through the
π
-stacked base pairs
within the helix.
23
Subtle perturbations to the DNA base stack,
including the presence of base pair mismatches, abasic sites, or
even DNA lesions, such as those that are substrates for DNA
glycosylases, attenuate DNA CT.
18,24
Protein binding can also
interrupt DNA CT if it disrupts base stacking, as seen with
enzymes that
fl
ip DNA bases out of the helix.
25
This CT
chemistry has been used to develop electrochemical sensors
that detect base lesions, mismatches, and DNA-binding
proteins on DNA-modi
fi
ed electrodes.
18,25
−
27
Charge can be
transported through DNA over long molecular distances, and
Received:
February 25, 2014
Published:
April 16, 2014
Article
pubs.acs.org/JACS
© 2014 American Chemical Society
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−
6478
the distance dependence of CT is quite shallow.
23
In fact,
charge can be e
ffi
ciently transported through at least 100 base
pairs, and over this distance the rate is still limited by transport
through the linker rather than the DNA base stack.
28,29
Given
that DNA CT can occur over long molecular distances and can
be modulated by DNA-binding proteins, does DNA-mediated
CT play a general role within the cell?
Recently DinG, a DNA damage response helicase from
E.
coli
, was shown to contain a 4Fe
−
4S cluster.
30
DinG is part of
the SOS response, which is activated by DNA damaging agents
and cellular stressors. DinG shares homology with the
nucleotide excision repair protein XPD as well as with a host
of Superfamily 2 helicases from archaea and eukaryotes that are
linked to human disease and share a conserved 4Fe
−
4S
domain.
5
DinG unwinds DNA that has single-stranded
overhangs with a 5
′
to 3
′
polarity.
31
DNA
−
RNA hybrid
duplexes that form within a DNA bubble, termed R-loops,
represent a unique substrate that DinG has been shown to
unwind
in vitro
.
32
Importantly, DinG is required to unwind R-
loops
in vivo
in order to resolve stalled replication forks and
thus to maintain the integrity of the genome.
33
Here we
examine the DNA-bound redox properties of DinG and explore
more generally crosstalk among redox-active DNA processing
enzymes in
E. coli
via 4Fe
−
4S clusters.
■
EXPERIMENTAL METHODS
Expression and Puri
fi
cation of DinG.
The
dinG
gene was
ampli
fi
ed from
E. coli
and was inserted into a pET-28 b (+) vector
(Novagen) as described previously.
30
After the vector was isolated, the
cloned
dinG
gene was sequenced (Laragen) using the primers listed in
Table S1. An aliquot of BL21(DE3) competent cells (Invitrogen) was
then transformed with the pET28b-
dinG
vector. The constructed
pET28b-
dinG
vector encodes for DinG with a C-terminal
hexahistidine a
ffi
nity tag.
To express DinG, 6 L of LB, which had been inoculated with an
overnight culture of BL21(DE3) cells harboring the pET28b-
dinG
vector, was shaken at 37
°
C. After the cultures reached an optical
density of
∼
0.6
−
0.8, enough IPTG (Research Products International
Corp.) was added to bring the concentration of IPTG in each
fl
ask to
150
μ
M. The
fl
asks were then returned to the incubator, which had
been cooled to
∼
22
°
C. After
∼
16 h of IPTG induction at
∼
22
°
C, the
cells were collected by centrifugation at 5500 rpm for 15 min. The cell
pellets were frozen at
−
80
°
C.
To purify DinG, the cell pellets were resuspended in 300 mL bu
ff
er
A (20 mM Tris-HCl, 8.0 pH at 4
°
C, 0.5 M NaCl, and 20% glycerol)
with added DNaseI from bovine pancreas (10 kU, Sigma) and
complete protease inhibitor cocktail tablets (Roche). The cells were
lysed using micro
fl
uidization. The lysate was centrifuged at 12 000 rpm
for 45 min, and the supernatant from the cell lysate was
fi
ltered and
loaded onto a 5 mL Histrap HP (GE healthcare) nickel-a
ffi
nity
column that had been equilibrated with bu
ff
er A. The column was then
connected to an A
̈
KTA fast protein liquid chromatography (GE
Healthcare) and was washed with 3
−
5 column volumes (CV) of bu
ff
er
A. The protein was eluted using a linear gradient from 0 to 20% bu
ff
er
B (20 mM Tris-HCl, 8.0 pH at 4
°
C, 0.5 M NaCl, 500 mM imidazole,
and 20% glycerol) over 10 CV, followed by a linear gradient from 20
to 30% bu
ff
er B over 10 CV. Fractions containing the desired protein,
which were yellow and eluted at
∼
150 mM imidazole, were desalted
into bu
ff
er C using a Hiprep 26/10 desalting column (GE Healthcare).
The collected protein was then concentrated down to 10
−
13 mL
using an Amicon Ultra-15 centri
fi
gual
fi
lter unit (Millipore) and was
loaded onto a Hiload Superdex 200 26/600 pg (GE healthcare) that
had been equilibrated with bu
ff
er C. The protein eluted after
∼
180 mL
of bu
ff
er C (20 mM Tris-HCl, 8.0 pH at 25
°
C, 0.5 M NaCl, and 20%
glycerol) had passed over the column. The purity of the protein was
con
fi
rmed using SDS-PAGE (Figure S1). A helicase activity assay for
DinG, modi
fi
ed from previously published procedures, was used to
show that the protein is active after puri
fi
cation.
30,32
DNA-Modi
fi
ed DinG Electrochemistry.
The DNA substrate
used for the electrochemical characterization of DinG was either a
well-matched 20-mer DNA oligomer with a 15-mer 5
′
to 3
′
single-
stranded overhang or the same substrate with the exception of an
abasic site being placed on the complementary strand four base pairs
from the bottom of the duplex (Table S1). A 20-mer strand of DNA
with a terminal thiol and 6-carbon linker at the 5
′
end of the strand
was annealed to a 35-mer unmodi
fi
ed strand of DNA to yield the
electrochemical substrate. The electrochemical substrate was designed
to be competent to be unwound by DinG in a helicase reaction. Single-
stranded DNA stimulates the ATPase activity of DinG, which requires
at least a 15-mer single-stranded 5
′
to 3
′
overhang in order to unwind
DNA substrates
in vitro.
32
In the electrochemical cell, the DNA
substrate is covalently tethered to the gold surface via a gold
−
thiol
bond.
The thiol-modi
fi
ed strand was synthesized on a 3400 Applied
Biosystems DNA synthesizer using standard phosphoramidite
chemistry. The complementary strands were purchased from IDT.
All phosphoramidites, including the terminal phosphoramidite
containing a 6-carbon disul
fi
de linker, were purchased from Glen
Research. The thiol-modi
fi
ed and complementary strands were
puri
fi
ed by HPLC using an analytical C-18 column (Agilent). DNA
strands were characterized by MALDI mass spectroscopy. The DNA
was quanti
fi
ed by UV
−
vis absorbance, and equimolar amounts were
annealed yielding the duplex substrate.
To prepare DNA-modi
fi
ed single electrodes, a 50
μ
M solution of
the DNA substrate was incubated overnight at ambient temperature
on a bare gold on mica surface (Agilent) in an electrochemical cell
with a capacity of 50
μ
L. Following incubation with the DNA solution,
the surface was rinsed and back
fi
lled by incubating the electrode with 1
mM 6-mercapto-1-hexanol for 45 min at room temperature. Multiplex
chip electrodes were prepared as described previously.
29,34
The well-
matched electrochemistry substrate was used for all single electrode
experiments. For experiments with multiplex chip electrodes, the well-
matched and abasic-site substrates were laid down side-by-side in
separate quadrants on a single chip.
29,34
After back
fi
lling, the DNA-modi
fi
ed electrodes were rinsed with the
electrochemistry bu
ff
er (4 mM spermidine, 4 mM MgCl
2
, 0.25 mM
EDTA, 20% glycerol, 250 mM NaCl, 20 mM Tris-HCl, pH
∼
8.5).
Protein concentration was measured by UV
−
vis absorbance using an
extinction coe
ffi
cient at 410 nm of 17 000 M
−
1
cm
−
1
.
22
An aliquot of
20
μ
M DinG was
fl
ash thawed by incubating it in a room temperature
water bath. The protein
’
sbu
ff
er was exchanged for the electro-
chemistry bu
ff
er by diluting the protein two-fold into 2
×
spermidine
bu
ff
er (8 mM spermidine, 8 mM MgCl
2
, 1 mM EDTA, 20 mM Tris-
HCl, pH
∼
9.0).
Electrochemical measurements were made using a CHI620D
Electrochemical Analyzer. For cyclic voltammetry, sweeps within a
window from
−
0.4 V vs Ag/AgCl to 0.1 or 0.2 V were carried out at a
scan rate of 50 mV/s for several hours. For electrochemistry
measurements on single electrodes with ATP, 1 mM ATP or 1 mM
ATP
γ
S (Sigma) was added after the electrochemical signal grew in to
an appreciable size (>20 nA). Cyclic voltammetry was then used to
scan the electrode over several hours.
Atomic Force Microscopy (AFM) Redistribution Assay.
AFM
experiments were performed using a protocol similar to that reported
previously with the following modi
fi
cations.
22,35
The long and short
strands of DNA have an identical sequence as they were both
ampli
fi
ed o
ff
of the pUC19 plasmid with primers containing a 2
′
O
-
methyl residue to generate 1.8 and 2.2 kb strands of DNA with 14-mer
single-stranded overhangs, so that the two could be subsequently
ligated. For well-matched long strands of DNA, the two PCR products
were annealed with complementary 14 bp overhangs. For the
mismatched long strands of DNA, the strands were annealed in the
same way except one of the PCR products contained a 14 bp overhang
with a single base changed to yield a C:A mismatch upon the annealing
of the two strands. Prior to deposition, the protein and DNA solution
was incubated at 4
°
C for 2 h. The sample was then deposited (5
−
10
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μ
L) onto a freshly cleaved mica surface for 2 min, rinsed with 2 mL of
water, and dried under argon. The concentration of DinG was 60 nM
for AFM experiments with DinG. For AFM experiments with mixtures
of DinG and EndoIII or DinG and EndoIII Y82A, the concentration of
each protein was 30 nM.
Images of protein and DNA mixtures that had been deposited on
dry mica surfaces were gathered using a Bruker Dimension Icon AFM
(Beckman Institute MMRC). Images were captured in air with scan
areas of 2
×
2or3
×
3
μ
m
2
in soft tapping mode and a scan rate of
3.00 Hz. RFESPA silicon AFM probes with re
fl
ective aluminum
backing (Bruker), with a spring constant of 3 N/m and a resonance
frequency of 69
−
81 kHz, were used for gathering images.
Bruker nanoscope analysis software was used to measure general
DNA contour lengths and height pro
fi
les of the proteins. For each data
set, images from at least three independently prepared surfaces were
analyzed. At least 50 images were analyzed for both the mismatched
DNA
−
protein samples and the well-matched samples. The binding
density ratio,
r
,isde
fi
ned as the ratio of the density of proteins bound
on the long strands of DNA divided by the density of proteins bound
on the short strands of DNA. The density of proteins on each strand is
found by dividing the number of proteins by the length of the DNA
strands, 3.8 kb pairs for the long strand and 1.9 kb pairs, which is the
average length of the short strands, for short strands. Error represents
SEM (
n
≥
3) for each experiment. Distinguishable strands and bound
proteins were counted by hand. In order to control for bias, for each
experiment, images were randomly assigned identi
fi
cation numbers.
The images were then scored blindly. The numbers of long strands,
proteins on long strands, short strands, and proteins on short strands
were collected for each image.
Binding density ratios can also be calculated for each individual
image, which are treated as replicates, to obtain the average binding
density ratio for each sample. The binding density ratios were plotted
as a histogram (Figure S2), which show that the binding density ratios
for the two sets of data follow a normal distribution around the mean,
allowing for statistical analysis with a two-tailed
t
test. Binding density
ratios obtained using this m
ethodology are presented in the
Supporting Information
MutY Activity Assay (CC104
lac
+
Forward Reversion Assay).
The method used to inactivate
dinG
on the genome within the CC104
strain was adapted from a previously published procedure.
36
The FRT-
fl
anked chloramphenicol acetyltransferase gene,
cat
, from pKD3 was
ampli
fi
ed by PCR using the
Δ
dinG
::cm
R
forward primer and
Δ
dinG
::cm
R
reverse primer (Table S1). Following inactivation of
dinG
in the CC104 strain, colony PCR with the
Δ
dinG
::cm
R
forward
and reverse sequencing primers (Table S1) was performed to con
fi
rm
dinG
had been replaced by
cat
. All PCR products were sequenced to
con
fi
rm that the chromosomal gene disruption was successful
(Laragen).
All mutant EndoIII plasmids, which were derived from
pBBR1MCS-4,
37
were generated using a QuikChange II Site-Directed
Mutagenesis kit (Agilent). The pBBR1MCS-4, pBBR1MCS-4-
nth
,
pBBR1MCS-4-
nth
D138A, and pBBR1MCS-4-
nth
Y82A plasmids
encode and constitutively express no protein, WT EndoIII, EndoIII
D138A, and EndoIII Y82A, respectively. The pBBR1MCS-4 derived
plasmids are referred to as p(empty), p(WT EndoIII), p(EndoIII
D138A), and p(EndoIII Y82A), respectively, throughout the text as
indicated in Table S2. The p(WT EndoIII) plasmid that was used as
the template for the site-directed mutagenesis reactions was previously
constructed in our laboratory.
35
The primers outlined in Table S1
were used to make the p(EndoIII Y82A) and p(EndoIII D138A)
mutant plasmids. The isolated plasmids were sequenced (Laragen)
using the forward and reverse pBBR1MCS-4-
nth
sequencing primers
(Table S1) to verify that the desired mutation had been made in the
nth
gene.
The MutY activity assay was adapted from a previously published
procedure.
22,38
The CC104 or CC104
Δ
dinG
::cm
R
strains were
transformed by electroporation with the p(EndoIII D138A), p-
(empty), and p(EndoIII Y82A) plasmids (Table S2). Following
transformation, the cells were recovered in 1 mL LB for 2 h. The cells
were spread on LB ampicillin (100
μ
g/mL) agar plates and incubated
for 16 h, after which a single transformant was restreaked onto a fresh
LB ampicillin (100
μ
g/mL) agar plate. These plates were incubated at
37
°
C for 12 h.
One colony from each strain was used to inoculate a 1 mL LB
ampicillin (100
μ
g/mL) culture. These cultures were incubated with
shaking at 37
°
C for 16 h. Next, 250
μ
L of each culture was spread
onto NCE lactose (0.2% w/v) ampicillin (40
μ
g/mL) agar plates and
incubated at 37
°
C. The number of colony-forming units, representing
lac
+
revertants, was counted after 48 h. Di
ff
erences for growth between
strains were monitored by evaluating the number of colony forming
units on NCE glucose (0.2% w/v) ampicillin (40
μ
g/mL) agar plates.
Note that we assay for MutY activity rather than EndoIII since the
frequency of spontaneous GC:TA transversions associated with 8-
oxoG:A repair by MutY is far greater than the pyrimidine lesions
repaired by EndoIII.
InvA
Δ
nth
Growth Assay.
The genomic
nth
gene, encoding
EndoIII, was knocked out of the InvA strain using P1 phage
transduction.
39
The JW1625
−
1
E. coli
strain from the Keio collection
was used as a donor strain to transduce the
nth
::kan
R
marker into an
E.
coli
strain, BW16847, which is an
E. coli
MG1655 derivative that
contains a Tn10 genomic marker near or within the
purR
gene, which
is around 26 kb downstream of the
nth
gene (Table S3). Both the
nth
::kan
R
and Tn10 markers from this newly generated strain (MG001
in Table S3) were transduced into the InvA strain, allowing for
isolation of the
nth
knockout in InvA (InvA
Δ
nth
) by selection on
tetracycline LB agar plates followed by veri
fi
cation of the disruption
using colony PCR with the
nth
genomic check primers (Table S1).
Plasmids that constitutively express various mutants of EndoIII were
prepared as outlined in the MutY activity assay section above. The
EndoIII plasmids p(WT EndoIII), p(EndoIII D138A), p(EndoIII
Y82A), and p(empty), in addition to an RNaseH overexpression
plasmid (Table S2) designated p(RNaseH), were transformed into the
InvA
Δ
nth
strain using standard electroporation techniques. Colony
PCR using the InvA check 1 and check 2 primers (Table S1) was used
to verify that InvA-derived strains still contained the inverted
rrnA
operon after these genetic manipulations and transformations. Colony
PCR with the genomic
nth
check primers (Table S1) was used to
verify that the
nth
gene was knocked out of each putative
Δ
nth
strain.
Growth curves of the InvA
Δ
nth
strain in addition to InvA
Δ
nth
transformed with p(WT EndoIII), p(EndoIII Y82A), p(EndoIII
D138A), p(empty), or p(RNaseH) were used to assess the e
ff
ect of
knocking out the
nth
gene from the InvA background. Single colonies
from LB ampicillin (100
μ
g/mL), or LB plates for each of the strains
were used to inoculate separate cultures of LB, LB ampicillin (100
μ
g/
mL), MM (M9 + 0.2% glucose),
40
or MM ampicillin (100
μ
g/mL).
Cell growth was then monitored through measurement of the optical
density at 600 nm for each of the cultures over time in LB or MM.
■
RESULTS
DNA Binding Activates DinG toward Reduction and
Oxidation at Cellular Redox Potentials.
DNA-modi
fi
ed
electrodes were utilized to explore the DNA-bound redox
chemistry of DinG. Cyclic voltammetry of the protein on gold
electrodes modi
fi
ed with a 20-mer duplexed DNA oligomer
appended with a 15-base 5
′
single-stranded overhang displays a
reversible redox potential for DinG of 80 mV vs NHE (Figure
1). This DNA-bound potential di
ff
ers from the midpoint redox
potential of
∼−
390 mV vs NHE assigned to the [4Fe
−
4S]
2+/1+
couple of the cluster observed in the absence of DNA as
measured by titrations with redox mediators.
30
Cyclic
voltammetry of DinG on multiplexed electrodes reveals that a
single abasic site placed in the DNA duplex attenuates the
current by 12
±
3%, consistent with the signal being DNA
mediated.
23,34
Moreover, upon addition of ATP to DinG bound
to DNA-modi
fi
ed electrodes, the reductive and oxidative peak
currents markedly increase; ATP
γ
S, which is poorly hydrolyzed,
does not yield a signi
fi
cant increase in current (Figure 1). Thus,
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it appears that the ATPase activity of DinG can be monitored
electrically, even though ATP hydrolysis is a redox-independent
process. Similar results were seen earlier with
S. acidocaldarius
XPD (SaXPD), also an ATP-dependent helicase.
21
It is
interesting to consider that this electronic signaling of activity
may be used within a biological context.
EndoIII and DinG use DNA CT to Redistribute to Sites
of DNA Damage.
Given that DinG displays a DNA-bound
potential similar to that of EndoIII of
∼
80 mV vs NHE, we
sought to test whether EndoIII and DinG can signal to one
another via DNA CT
in vitro
to aid one another in
fi
nding
lesions that disrupt CT. It is noteworthy that both repair
proteins are involved in
fi
nding lesions that interrupt DNA CT.
Using AFM, we examined whether the DinG helicase would
redistribute onto 3.8 kilobase (kb) DNA strands containing a
single base mismatch, which interrupts DNA CT, rather than
remaining bound to well-matched DNA strands. Our model for
how repair proteins utilize DNA CT predicts such a
redistribution as a
fi
rst step in repair (
vide infra
), and this
assay provides direct support for the model. If proteins of
similar potential carry out DNA CT on well-matched DNA
strands and dissociate from DNA upon reduction, they should
preferentially redistribute onto DNA strands where DNA CT is
inhibited by an intervening mismatch. Note that, while a single
base mismatch inhibits DNA CT, it is not a substrate for either
DinG or EndoIII binding. We have utilized this AFM assay
previously to test EndoIII redistribution as a
fi
rst step in
fi
nding
damage.
22
We have also utilized this assay to test CT signaling
between EndoIII and SaXPD, which also contains a 4Fe
−
4S
cluster with a DNA-bound potential of
∼
80 mV vs NHE, in
locating DNA damage;
21,35
these proteins are present in
completely distinct organisms, but based on their shared DNA-
bound potential are able to signal one another using DNA CT.
In this AFM assay, DNA
−
protein mixtures are deposited
onto a dry mica surface on which single molecules of both free
and protein-bound DNA can be visualized.
22,35
Duplexes of
DNA that contain a single C:A mismatch located in the middle
of the strand are mixed with fully matched DNA. These strands
can be distinguished in the AFM by their di
ff
erence in length:
the mismatched strands are
∼
3.8 kb pairs long, while the
matched strands on average contain
∼
1.9 kb pairs (Figure 2).
Figure 1.
Electrochemistry of DinG on DNA-modi
fi
ed electrodes. (A)
Cyclic voltammogram of 10
μ
M DinG (red), DinG after the addition
of 5 mM ATP (blue), and bu
ff
er only (black) after incubation for 3 h.
Inset: Cartoon representation of a protein bound to DNA on a DNA-
modi
fi
ed electrode. (B) Percent change in current after the addition of
1 mM ATP (blue) or 1 mM ATP
γ
s (black). Percent change in current
is the percent increase in the measured current compared to the
predicted current, based on the linear growth of the signal with respect
to time for the incubation of DinG before the addition of ATP.
Figure 2.
AFM redistribution assay. (A) A
fl
attened image (Bruker
nanoscope analysis software) for tapping-mode AFM topography of
DinG-bound DNA adsorbed on mica. (B) Schematic representation of
the redistribution assay. At equilibrium, repair proteins (blue) are
preferentially localized on strands of DNA (black) with a C:A
mismatch (red X). (C) Three-dimensional rendering of the blue-
bordered region of the AFM image in A that shows a strand of DNA
bound by two DinG proteins. (D) Measured binding density ratios,
the density of proteins on long strands divided by the density of
proteins on short strands, for proteins bound to mixtures of long and
short strands of DNA with and without a mismatch (C:A) in the
middle of the long strand. Three separate mixtures of protein and
DNA were deposited onto individual surfaces, and at least 50 images
were analyzed for each DinG (blue), a mixture of DinG and EndoIII
(red), and a mixture of DinG and a CT-de
fi
cient mutant, Y82A
EndoIII (green).
±
SEM using a single image as a data point.
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They share DNA sequence since the 3.8 kb strands are
prepared by ligation of the two shorter strands.
22,35
For
mixtures of mismatched long strands and well-matched short
strands that are incubated with DinG alone, we
fi
nd an average
of 2.60
±
0.22 proteins bound per mismatched strand
compared to 0.90
±
0.17 proteins per well-matched strand.
We calculate the binding density on the mismatched and
matched strands for each independent trial (
n
≥
3 for each
experiment) by normalizing the number of proteins bound by
the strand length to obtain a binding density ratio of 1.44
±
0.08 favoring the mismatched strand. Thus, even though DinG
does not bind preferentially to a mismatch, DNA CT by DinG
favors its redistribution onto the strand containing the single
base mismatch.
Signaling between EndoIII and DinG was also tested by
AFM. In 1:1 mixtures of DinG and wild-type (WT) EndoIII, a
binding density ratio of 1.32
±
0.04 favoring the mismatched
strand is observed (Figure 2). But does this redistribution
depend upon DNA CT? In 1:1 mixtures of DinG and EndoIII
Y82A, a mutant protein that is defective in DNA-mediated
CT,
22,35
a binding density ratio of 0.90
±
0.03 is found (Figure
2); there is no preference for the mismatched strand (note that
EndoIII does not bind preferentially to a mismatch).
22
This
binding density ratio is comparable to what is observed for
DinG alone when both strands are fully matched (Figure 2);
when the proteins cannot carry out DNA CT, they cannot
redistribute onto the strand containing the lesion. Since DinG
can redistribute in the absence of EndoIII and DinG and
EndoIII can redistribute when mixed only if EndoIII is e
ff
ective
in signaling by DNA CT, these observations support the need
for e
ff
ective signaling between EndoIII and DinG in
fi
nding the
damaged strand. We have seen comparable results earlier in
mixtures of SaXPD and EndoIII.
35
It should be noted that protein loadings on the 3.8 kb strands
are on the order of two proteins per strand under these
experimental conditions. Therefore, assuming that DinG and
EndoIII are signaling one another, for approximately half of the
strands, signaling must occur between DinG and EndoIII rather
than just between DinG partners or between EndoIII partners.
Moreover, given a loading of about two proteins per strand,
these results are consistent with DNA CT occurring over
kilobase distances. It is also important to note that these
proteins show no evidence of colocalizing at DNA sites by
AFM. Overall, these data demonstrate that DinG and EndoIII
can use DNA-mediated CT at long range to cooperate with one
another to localize to regions of damage.
DinG Uses DNA-Mediated CT to Facilitate the Repair
of DNA Damage by MutY.
To begin to probe DNA-
mediated signaling within the cell, a
lac
+
forward reversion assay
reporting on GC:TA transversions that re
fl
ects MutY activity
within the CC104 strain of
E. coli
was utilized.
38
The CC104
strain reports on GC to TA transversions, which are prevented
by MutY excising adenines improperly placed opposite an 8-
oxodG lesion, in the lacZ gene. Cells in which this transversion
process has occurred form colonies on plates containing lactose
as the sole carbon source and are termed
lac
+
revertants. Using
this strain, changes in MutY activity can be assessed upon
genetically knocking out DinG. If DinG and MutY cooperate in
fi
nding DNA lesions that attenuate CT, eliminating DinG from
the cell should lead to a decrease in MutY activity and a
corresponding increase in
lac
+
revertants. We and others had
earlier seen an e
ff
ect of knocking out EndoIII on MutY activity
in this assay.
22,41
When
dinG
is knocked out of the CC104
strain (CC104
Δ
dinG
), we
fi
nd that the number of revertants
increases 60% compared to WT CC104 (Table 1). While the
e
ff
ect is not high, it is notable given that knocking out MutY
itself gives a maximum of 10
−
15 fold increase in revertant
count under similar conditions.
22
It should also be noted that
EndoIII and DinG do not have overlapping substrate speci
fi
city
with MutY. A deletion of the
nth
gene from CC104 in
combination with a deletion of the
mutY
gene resulted in the
same number of revertants as that of the
mutY
deletion alone.
22
If the e
ff
ect on MutY activity stems from lowering the
concentration of DNA-bound 4Fe
−
4S proteins that coopera-
tively signal with MutY, then complementing the cells with a
plasmid that expresses a mutant of EndoIII that contains a
4Fe
−
4S cluster, but is enzymatically inactive, p(EndoIII
D138A) (Table 1),
22,42
should rescue MutY activity. Indeed,
complementation with the plasmid for EndoIII D138A restores
the activity of MutY in CC104
Δ
dinG
; the number of
revertants found is comparable to WT CC104. However,
complementing with a plasmid that expresses an EndoIII
mutant, p(EndoIII Y82A), which is defective in DNA CT but
nonetheless contains a 4Fe
−
4S cluster and is enzymatically
active,
22
does not rescue MutY activity (Table 1). It should be
notedthatrescuewithDinGwasnotexplored,since
overexpression of DinG using plasmids was previously observed
to be toxic to cells.
33
While these data may not indicate
dramatic e
ff
ects, they are statistically signi
fi
cant and fully
consistent with our model. These data thus suggest that MutY,
EndoIII, and DinG may be capable of signaling one another via
DNA-mediated CT to coordinate their activity in cells.
Repair of R-Loops by DinG Relies on DNA-Mediated
Signaling by EndoIII in InvA.
Perhaps more interesting to
consider is the possibility of signaling in the reverse sense, with
a BER protein signaling to DinG to aid DinG in
fi
nding its
lesions, and here dramatic e
ff
ects on survival are observed.
Does EndoIII signaling aid DinG in locating R-loops, a
substrate of DinG, in
E. coli
? R-loops are RNA:DNA hybrids
that perturb duplex base stacking and would be expected to
attenuate DNA CT, so that, based on the AFM results, DNA-
mediated signaling could help DinG in its
fi
rst step of
fi
nding
its substrate. Here we took advantage of a strain, InvA, where
the repair of R-loops by DinG is critical for cell survival. In the
InvA strain, by inverting a frequently transcribed
rrnA
operon,
the incidence of R-loop formation in cells is increased;
33
as a
Table 1. MutY Activity Assay (CC104
lac
+
Forward
Reversion Assay)
strain
plasmid
a
no. of
lac
+
revertants
b
relative to
CC104
c
CC104
p(empty)
4.6
±
0.4
1
CC104
Δ
dinG
p(empty)
7.5
±
0.7
1.6
d
CC104
p(EndoIII
D138A)
8.5
±
1.1
1
CC104
Δ
dinG
p(EndoIII
D138A)
6.7
±
0.6
0.8
d
CC104
p(EndoIII Y82A)
3.4
±
0.3
1
CC104
Δ
dinG
p(EndoIII Y82A)
5.8
±
0.8
1.7
d
a
See Table S2 for full plasmid designation.
b
Values for
lac
+
revertants
per 10
9
cells are reported for at least three independent trials (
N
≥
20)
±
SEM.
c
Relative to CC104 is de
fi
ned as the ratio of
lac
+
revertants for
the CC104
dinG
knockout strain to the number of
lac
+
revertants for
the wild-type CC104 strain containing the same plasmid as the CC104
dinG
knockout strain
d
(
p
< 0.01)
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result, replication forks stalling, when they collide head-on with
the transcription machinery, are necessarily ampli
fi
ed in the
InvA strain. Resolving stalled replication forks is vital to
preventing signi
fi
cantly deleterious DNA damage and thus to
survival.
33,43
In previous work it has been shown that within the
InvA strain, the repair of R-loops by DinG was shown to be
essential to maintain viability; deletion of
dinG
yielded a
signi
fi
cant plating defect.
33
Rescue by overexpression of
RNaseH is a hallmark of R-loop-dependent phenotypes, as
RNaseH endonucleolytically degrades RNA within an
RNA:DNA hybrid but not free RNA.
33
Complementing the
InvA
Δ
dinG
strain with a plasmid encoding RNaseH thus
rescued activity.
33
Growth assays to test DinG activity using
InvA are particularly advantageous in testing signaling with
other proteins since the growth defect is dramatic; knocking
out signaling partners should similarly yield clearly discernible
e
ff
ects.
To test signaling between EndoIII and DinG, we prepared an
EndoIII knockout in the InvA strain (InvA
Δ
nth
). If signaling
with EndoIII is essential for DinG to e
ff
ectively repair R-loops,
consistent with our model, we would expect to observe e
ff
ects
on cell viability upon knocking out the
nth
gene in InvA. After
incubation in LB for several hours, growth of the InvA
Δ
nth
strain is indeed compromised compared to the InvA parent
strain (Figure 3). If the strains are instead grown under nutrient
deprivation in minimal media, cellular viability is completely
lost (Figure S3). The growth curves for InvA
Δ
nth
alongside
InvA or InvA
Δ
nth
strains with transformed plasmids that
express various EndoIII mutants and RNaseH are shown in
Figure 3. If the growth defect for InvA
Δ
nth
is the result of the
ine
ff
ective repair of R-loops, as was seen for the InvA
Δ
dinG
phenotype, complementation with a plasmid that overexpresses
RNaseH, p(RNaseH), should restore activity, which is
observed. This result indicates signaling between EndoIII and
DinG but does not elucidate the mechanism of signaling. The
cooperative signaling e
ff
ect is not due to the enzymatic activity
of EndoIII. The p(EndoIII D138A) plasmid, which expresses
the EndoIII mutant lacking glycosylase activity but containing a
4Fe
−
4S cluster, also rescues the InvA
Δ
nth
strain. But is this
cooperative signaling the result of long-range DNA CT among
4Fe
−
4S proteins? While expressing WT EndoIII restores
activity in InvA
Δ
nth
, complementation with the CT-defective,
but enzymatically pro
fi
cient p(EndoIII Y82A) plasmid does
not. These results strongly correlate with the results from the
AFM and reversion assays, and it appears that EndoIII signals
to DinG via DNA CT to help DinG locate and process R-loops.
■
DISCUSSION
Our model for how DNA repair proteins with 4Fe
−
4S clusters
use DNA-mediated CT as a
fi
rst step in locating lesions to
repair is depicted in Figure 4. Critical to the model is the fact
that the DNA binding a
ffi
nity of a protein that has a 4Fe
−
4S
cluster is dependent on the oxidation state of the cluster. For
these proteins, the shift in reduction potential upon DNA
binding necessitates a lower DNA binding a
ffi
nity of at least 3
orders of magnitude for a 200 mV shift when in the reduced
form (2+) compared to the oxidized form (3+).
19
Figure 1
shows that the DNA-bound potential is signi
fi
cantly shifted
from that reported in the absence of DNA.
30
Therefore, as
illustrated in Figure 4, 4Fe
−
4S clusters in these proteins when
Figure 3.
Rescue of growth defect conferred by knocking out
nth
in InvA. Cultures of LB were inoculated with single colonies of each strain and
growth was monitored by optical density at 600 nm over time. Strains of InvA
Δ
nth
grew comparably to InvA
Δ
nth
transformed with p(empty)
showing that the e
ff
ect is not due to the presence of the plasmid. Data were recorded for at least three independent trials. (A) Growth of InvA WT
(blue) or InvA
Δ
nth
transformed with p(empty) (red)
±
SEM. (B) Growth of InvA
Δ
nth
transformed with p(WT EndoIII) (blue) or p(empty)
(red)
±
SEM. (C) Growth of InvA
Δ
nth
transformed with p(RNaseH) (blue) or p(empty) (red)
±
SEM. (D) Growth of InvA
Δ
nth
transformed
with p(EndoIII D138A) (blue) or p(empty) (red)
±
SEM. (E) Growth of InvA
Δ
nth
transformed with p(EndoIII Y82A) (black) or p(empty) (red)
±
SEM.
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they are freely di
ff
using are expected to be in the 2+ state.
Upon binding to DNA, however, the proteins are activated
towards oxidation. A given protein already bound to DNA in
the oxidized form, perhaps oxidized from a distance by a
guanine radical generated under oxidative stress,
44
could thus
be reduced in a DNA-mediated fashion by another distinct
redox-active protein that binds within CT distance of the
fi
rst
protein. Reducing this second protein would promote its
dissociation from DNA. This interprotein signaling requires an
undamaged path between the two proteins; intervening DNA
damage prevents the protein from receiving reducing
equivalents so that its dissociation is not promoted. E
ff
ectively,
this electron-transfer event signals the repair protein to
dissociate from undamaged regions and search for damage
elsewhere in the genome. If there is an intervening damage
product that blocks CT, however, then the repair protein stays
bound in the vicinity of damage, and the protein can move on a
slower time scale to the local site in need of repair. This process
would lead to the redistribution of repair proteins in the vicinity
of damage through an e
ffi
cient scanning of the genome by
proteins of similar redox potential. In essence, these proteins
inform one another about the integrity of DNA by using DNA
as a medium through which they transmit electronically
encoded information. Because this signaling occurs over long
distances, this mechanism would signi
fi
cantly reduce the time
required to scan the genome, allowing for enzymes to repair the
genome on biological time scales. Indeed, even when CT
distances of only 100 bases, that which we have documented,
are permitted in our model, a signi
fi
cant reduction in search
time to scan the
E. coli
genome can be predicted.
22
Importantly,
other models have been investigated for how BER enzymes
similar to MutY and EndoIII can scan the genome and locate
their substrates. For example, it has been shown that one-
dimensional sliding along DNA can be fast enough for
glycosylases to come into contact with bases in the genome
on the order of seconds.
45a
−
c
Models for one-dimensional
sliding do not, however, take into account protein tra
ffi
c along
the genome. It is important to note that DNA-mediated CT is
not interrupted by intervening bound proteins as long as the
proteins do not perturb the base pair stack. Thus, sliding,
hopping, and DNA CT models taken together o
ff
er an
appealing means to explain how the search process may be
optimized under the realistic conditions of the cell.
Data from DNA-modi
fi
ed electrochemistry experiments
show that the DNA-bound reduction potential of DinG is
remarkably similar to that for EndoIII, MutY, and SaXPD.
19
−
21
As such, DinG is competent to shuttle electrons through DNA
to or from EndoIII or MutY via its 4Fe
−
4S cluster, as would be
required by the model proposed for the redistribution of these
proteins to sites of damage. As with EndoIII and MutY, we
consider the redox potential of DinG to correspond to the
[4Fe
−
4S]
3+/2+
couple that is now accessible due to the negative
potential shift associated with binding to the DNA polyanion.
19
The ATP-dependent increase in current intensity observed for
DinG on electrodes is consistent with previous results for
SaXPD, except that the signal increase is nearly an order of
magnitude higher than that observed for the thermophilic
SaXPD.
21
This substantial di
ff
erence in signal increase is
understandable based upon the signi
fi
cantly lower rate of ATP
hydrolysis of SaXPD versus DinG at ambient temperature. It is
interesting to consider that the increase in signal intensity could
be a general characteristic of these DNA enzymes that contain
redox-active clusters, where they signal not only their presence
but also their activity. For DinG, there could be signaling to
upstream proteins that DinG is in the process of unwinding its
substrate.
The AFM experiments, moreover, support signaling between
EndoIII and DinG
in vitro
. Based on the model, we expect the
redistribution of proteins that use DNA-mediated CT signaling
onto strands containing a single base mismatch and away from
fully matched duplex DNA, which is the observed result.
Proteins that are defective in DNA CT, furthermore, do not
relocate to the mismatched strand, as predicted by our model.
Since R-loops disrupt DNA CT, it would be expected that this
chemistry could be used within a cell as a
fi
rst step to drive the
redistribution of DinG into the vicinity of R-loops as well; a
binding preference of DinG for the R-loop would lead to
subsequent localization.
Importantly, the genetics experiments point to a role for
DNA-mediated signaling by DinG and other proteins with
4Fe
−
4S clusters within the cell. Based on the hypothesis that
any DNA processing enzymes with 4Fe
−
4S clusters of similar
potential can cooperate, we would expect signaling between
EndoIII and DinG and also signaling between MutY and DinG.
The
lac
+
forward reversion assay demonstrates that DinG does
indeed help MutY
fi
nd and process its substrate, since knocking
out DinG results in an increase in mutagenesis associated with a
decrease in MutY activity. Moreover, expressing an enzymati-
cally de
fi
cient mutant of EndoIII still rescues activity, while a
CT-de
fi
cient mutant does not. Interestingly, this mutagenesis is
suppressed by expression of EndoIII, despite the fact that
EndoIII does not repair the same lesions as MutY.
22
It is
remarkable to consider that an e
ff
ect caused by knocking out a
helicase can be reversed by expressing a separate DNA
glycosylase, EndoIII. What they have in common is the 4Fe
−
Figure 4.
Scheme depicting how repair proteins may use DNA-
mediated signaling to search for damage. The model describes how
DNA CT can drive the redistribution of the repair proteins into the
vicinity of damage. (1) A protein with a reduced (orange-yellow)
iron
−
sulfur cluster binds to DNA. (2) This protein
’
s iron
−
sulfur
cluster is oxidized (purple-brown) by another DNA-bound redox-
active protein. This oxidation can occur over long distances and
through other DNA-bound proteins (gray) so long as the
π
-orbital
stacking of bases between the reductant and oxidant is unperturbed.
(3) Reduction promotes the repair protein
’
s dissociation from DNA.
(4) The repair protein binds to an alternate DNA site where it is
oxidized either by a guanine radical or another protein. (5) DNA
lesions between proteins inhibit electron transport, so protein
dissociation is not promoted. (6) Proteins that are now in close
proximity to the lesion are able to move processively toward the
damage for repair.
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4S cluster. These results provide genetic evidence that the e
ff
ect
of knocking out DinG is due to DinG aiding MutY in
processing its target lesion via DNA-mediated CT.
It is critical in the context of this model to demonstrate not
only that DinG a
ff
ects the activity of base excision repair
enzymes with 4Fe
−
4S clusters but also that base excision repair
enzymes a
ff
ect the activity of DinG. Within the InvA strain, R-
loop formation is signi
fi
cantly ampli
fi
ed, and the repair of R-
loops becomes essential for cell viability under certain
conditions. The fact that DinG is critically important in the
InvA strain is understandable. But we
fi
nd that by knocking out
even EndoIII in InvA, a dramatic growth defect in LB and a
complete loss of cellular viability under low nutrient conditions
is observed. Since RNaseH can compensate for a loss of DinG
activity by degrading R-loops, overexpression of RNaseH
restoring normal growth con
fi
rms that the observed growth
defect is due to EndoIII aiding DinG in processing R-loops. Just
as was seen in the
lac
+
forward reversion assay, expression of
the enzymatically inactive but CT-pro
fi
cient EndoIII D138A
can also restore normal growth. Expressing EndoIII Y82A,
which is CT-de
fi
cient, however, does not rescue growth.
Therefore, it is not the loss of glycolytic activity of EndoIII that
is suppressing growth in an EndoIII knockout in InvA but the
loss of the ability of EndoIII to carry out DNA-mediated CT
chemistry.
Overall these results provide substantial evidence, using both
AFM and genetics experiments, that
E. coli
enzymes from
distinct repair pathways signal one another from a distance
through DNA as long as the proteins remain competent to
carry out DNA-mediated CT, as measured electrochemically.
The AFM experiments show that a single base mismatch in a
3.8 kb duplex is su
ffi
cient to promote the redistribution of the
4Fe
−
4S proteins to damaged DNA, driven by long-range
signaling. The genetics experiments emphasize that cooperative
signaling for repair within the cell requires the ability of the
proteins with 4Fe
−
4S clusters to carry out DNA CT, not their
primary enzymatic activity. Signaling through DNA CT is fast
(ps),
46
can occur over long molecular distances, and allows for
the binding of many intervening proteins, as long as their
distortion of the DNA duplex is minimal. As such, DNA CT
provides a mechanism for e
ffi
cient signal transduction on
biological time scales, as the cell requires. Our proposed
redistribution model is one way in which proteins may use
DNA CT to e
ffi
ciently scan the genome as a
fi
rst step in
fi
nding
lesions to repair and to prepare the genome for replication. The
utilization of DNA CT by enzymes to maintain cellular viability
and genomic integrity represents a novel role for 4Fe
−
4S
clusters in DNA processing enzymes. A growing body of
evidence is emerging that highlights the importance of iron
−
sulfur clusters in enzymes that are involved in nearly every
aspect of DNA metabolism. The results here provide a basis for
understanding the ubiquity of 4Fe
−
4S clusters in proteins that
maintain the integrity of the genome throughout the
phylogeny.
■
ASSOCIATED CONTENT
*
S
Supporting Information
Experimental analysis and supporting
fi
gures and tables. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
jkbarton@caltech.edu
Notes
The authors declare no competing
fi
nancial interest.
■
ACKNOWLEDGMENTS
We are grateful to the NIH (GM49216), the Moore
Foundation (Caltech Signaling Center), and the Center for
Environmental Microbial Interactions (CEMI, Caltech) for
their
fi
nancial support. M.A.G. and H.M.S. were NIH
predoctoral trainees (NIH/NRSA 5T32GM07616) and T.J.Z.
an NSF fellow (DGE-1144469). We are also grateful to the
Beckman Institute MMRC for AFM instrumentation. We thank
Dr. Huangen Ding (Louisiana State University, Baton Rouge,
LA) for helpful discussions regarding DinG, the Coli Genetic
Stock Center (CGSC, Yale) for distributing the BW16847 and
JW1625-1, and Dr. B. Michel (Gif-sur-Yvette) for the gift of the
InvA bacterial strain and RNaseH overexpression plasmid.
Lastly we thank Ms. Janani Comar for research assistance.
■
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