of 8
E
ff
ective Distance for DNA-Mediated Charge Transport between
Repair Proteins
Edmund C. M. Tse,
Theodore J. Zwang,
Sebastian Bedoya, and Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
*
S
Supporting Information
ABSTRACT:
The stacked aromatic base pairs within the
DNA double helix facilitate charge transport down its length
in the absence of lesions, mismatches, and other stacking
perturbations. DNA repair proteins containing [4Fe4S]
clusters can take advantage of DNA charge transport (CT)
chemistry to scan the genome for mistakes more e
ffi
ciently.
Here we examine the e
ff
ective length over which charge can
be transported along DNA between these repair proteins. We
de
fi
ne the e
ff
ective CT distance as the length of DNA within
which two proteins are able to in
fl
uence their ensemble
a
ffi
nity to the DNA duplex via CT. Endonuclease III, a DNA repair glycosylase containing a [4Fe4S] cluster, was incubated with
DNA duplexes of di
ff
erent lengths (1.5
9 kb), and atomic force microscopy was used to quantify the binding of proteins to
these duplexes to determine how the relative protein a
ffi
nity changes with increasing DNA length. A sharp change in binding
slope is observed at 3509 base pairs, or about 1.2
μ
m, that supports the existence of two regimes for protein binding, one within
the range for DNA CT, one outside of the range for CT; DNA CT between the redox proteins bound to DNA e
ff
ectively
decreases the ensemble binding a
ffi
nity of oxidized and reduced proteins to DNA. Utilizing an Endonuclease III mutant Y82A,
which is defective in carrying out DNA CT, shows only one regime for protein binding. Decreasing the temperature to 4
°
Cor
including metallointercalators on the duplex, both of which should enhance base stacking and decrease DNA
fl
oppiness, leads to
extending the e
ff
ective length for DNA charge transport to
5300 bp or 1.8
μ
m. These results thus support DNA charge
transport between repair proteins over kilobase distances. The results furthermore highlight the ability of DNA repair proteins
to search the genome quickly and e
ffi
ciently using DNA charge transport chemistry.
INTRODUCTION
Cellular oxidative stress, external UV irradiation, and environ-
mental mutagens cause gen
omic lesions and base pair
modi
fi
cations in living organisms on the order of tens per
cell per second.
1
,
2
Various repair mechanisms are present in
biological systems to search and correct DNA lesions and
mismatches, thereby upholding genomic integrity.
3
,
4
It is
important that this repair be carried out quickly, before the
damage can in
fl
uence downstream processes or lead to
mutations.
5
,
6
In humans, accumulation of mutations caused
by defective DNA repair can lead to the proliferation of tumor
cells and development of cancer.
7
,
8
Recently, many DNA repair proteins have been found to
possess redox-active [4Fe4S] clusters that may expedite their
DNA damage search.
9
11
Experiments using DNA-modi
fi
ed
electrodes show that these proteins all share a broad DNA-
bound potential of
80 mV versus NHE, well below the
potential at which DNA bases are oxidized.
12
,
13
Although these
[4Fe4S] cluster repair proteins are typically present in cells in
relatively low copy numbers,
14
,
15
they must
fi
nd and repair
thousands of mutagenic lesions within the 20 min doubling
time of
Escherichia coli
.
16
,
17
Translocation is insu
ffi
cient to
explain the fast action of these proteins, especially when
accounting for limitations of di
ff
usion in the congested cellular
environment.
16
DNA-processing proteins containing [4Fe4S] clusters are
found in archaea, bacteria, and eukaryotes.
18
23
Primases and
polymerases containing [4Fe4S] clusters function as redox
switches that control DNA replication processes.
24
,
25
Base
excision repair proteins such as EndoIII, MutY, and uracil
DNA glycosylase (UDG), as well as SF2 helicases XPD, and
DinG contain redox-active [4Fe4S] clusters.
26
33
These
[4Fe4S] clusters may play a vital functional role in the DNA
damage search and repair scheme, though there is still much to
be understood about how they function in biological
systems.
34
37
Recent experiments with DNA-modi
fi
ed Au electrodes and
microscale thermophoresis (MST) suggest that the [4Fe4S]
cluster acts as a redox-modulated a
ffi
nity switch for DNA.
38
Using MST to quantify the binding a
ffi
nity of DNA to EndoIII,
EndoIII with an oxidized [4Fe4S]
3+
cluster was found to bind
DNA 550-fold stronger than that with a reduced [4Fe4S]
2+
cluster.
38
Electrochemical experiments also demonstrated that
binding to the anionic phosphate backbone of DNA shifts the
Received:
August 14, 2018
Published:
January 11, 2019
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redox potential of [4Fe4S] clusters in DNA repair proteins
negative by about 200 mV.
39
This shift in redox potential
corresponds to a change in the DNA-binding a
ffi
nity that is
similar to the values determined using MST.
40
The e
ff
ect of
binding to the polyanionic phosphate backbone of DNA
contributes substantially to the shift in redox potential of the
[4Fe4S] cluster in DNA repair proteins.
38
,
40
Previous experi-
ments have demonstrated that changing bu
ff
er salts result in
only small di
ff
erences in redox potential.
41
Atomic force
microscopy (AFM) experiments show that these proteins
reduce and oxidize one another through a DNA-mediated
redox signaling process, e
ff
ectively allowing for [4Fe4S] cluster
proteins to change one another
sa
ffi
nity to DNA.
11
,
38
We have proposed that these proteins containing [4Fe4S]
clusters utilize DNA charge transport (CT) as a means more
rapidly and e
ffi
ciently to search the genome for lesions. Using
ultrafast spectroscopy and electrochemical experiments, DNA
CT occurs rapidly through well stacked DNA in both the
excited and ground states but is interrupted by lesions,
mismatches, and other perturbations to the duplex base pair
stacking.
42
44
Redox-active [4Fe4S] cluster repair proteins can
utilize the disruption of DNA CT by lesions to screen the
genome for DNA damage.
45
,
46
Inside cells, oxidative stress may
oxidize the [4Fe4S] cluster of the proteins, or oxidize DNA
bases that can then undergo CT with the protein.
47
,
48
When
the proteins bind to the DNA polyanion, they are activated
toward oxidation. Hence, they are more prone to bind to DNA
and lose an electron in the process.
47
If there is no DNA
damage, the electron can travel down the length of DNA and
reduce the oxidized [4Fe4S]
3+
cluster of another repair protein,
thereby decreasing its a
ffi
nity and releasing it from DNA.
49
This released protein can then continue its search elsewhere.
48
This method, as a
fi
rst step in the search process, allows for
proteins to
fi
nd DNA damage much faster than by trans-
location alone.
50
Incorporating DNA-mediated charge trans-
port into the search, and assuming an e
ff
ective DNA CT length
of 200
500 bp between proteins, the time needed for [4Fe4S]
cluster repair proteins to search for DNA damage sites
shortens by at least an order of magnitude. By utilizing DNA
CT in the DNA damage search process, the time needed for
[4Fe4S] repair proteins to scan the entire genome is well
within the time constraints of other biological processes.
16
One
key advantage of a DNA CT supported search scheme is that
electrons can travel through DNA across a heavily congested
cellular environment in a relatively unhindered fashion on a
nanosecond time scale,
50
as opposed to a search scheme
utilizing only translocation by proteins that takes much longer
to cover the same distance, and can be made even slower than
0.1
10
μ
m
2
/s in
E. coli
because the crowded cellular
environment impedes movement.
16
,
51
53
However, what is the distance over which DNA CT can
proceed? Previously we have utilized a 100 bp dsDNA-
modi
fi
ed electrode to demonstrate that DNA CT can occur
over at least a distance of 34 nm.
54
Practical synthetic
limitations have, however, precluded the examination of longer
distances on electrodes, including those larger than the DNA
duplex persistence length of 50 nm (150 bp).
55
58
In addition,
extrapolation from experiments with shorter DNA on DNA-
modi
fi
ed electrodes has been inadequate for predicting a
maximum length because the rate of electron transfer is
generally limited by the attachment to the electrode.
54
Moreover, photophysical experiments varying the length of
DNA have shown an extremely shallow distance dependence
for DNA CT, while the same synthetic limitations apply.
54
,
59
AFM experiments allow for the interrogation under
equilibrium conditions of DNA CT distance over 100 times
longer than that used on DNA-modi
fi
ed electrodes, but cannot
elucidate the kinetics of this process.
11
,
38
After proteins and
DNA were allowed to interact, AFM is used to count the
number of proteins bound on DNA.
60
The AFM experimental
procedure does not change the population of proteins bound
on DNA.
16
Previous AFM experiments demonstrate that
DNA-mediated CT can occur between di
ff
erent proteins
containing [4Fe4S] clusters.
16
,
47
,
48
,
50
,
60
Using our AFM assay
to probe long-range signaling between proteins has, moreover,
suggested that DNA CT might proceed over kilobase
lengths.
11
Experimental data from these experiments suggest
that DNA CT lowers the e
ff
ective binding a
ffi
nity of any pair
of reduced and oxidized [4Fe4S] proteins relative to the
a
ffi
nity of a protein with oxidized [4Fe4S]
3+
that is not
involved in DNA CT.
38
Thus, if new proteins bind to DNA
with reduced [4Fe4S]
2+
clusters and are capable of DNA CT
with a bound protein containing an oxidized [4Fe4S]
3+
, the
e
ff
ective a
ffi
nity of these proteins will be decreased relative to
the oxidized [4Fe4S]
3+
protein alone.
If a maximum DNA CT length exists, we expect to detect
two regimes in AFM experiments with di
ff
erent observable
a
ffi
nities for DNA
protein interactions: shorter DNA where
all the proteins that bind are capable of DNA CT with another
protein, and longer DNA where proteins can bind outside of
maximum DNA CT distance of other proteins (
Figure 1
). In
the shorter regime all of the proteins have decreased a
ffi
nities
relative to a lone oxidized [4Fe4S]
3+
protein, but in the longer
regime many of the oxidized proteins will not have their a
ffi
nity
Figure 1.
Quantifying the DNA charge transport length. (a) Model showing the implications of DNA CT length on the redox signaling capability
between [4Fe4S] repair proteins and subsequently the lesion search process. (b) AFM images of two linear dsDNA. The one on the top is in the
regime where the DNA length is shorter than the CT length, while the one on the bottom is in the regime where the DNA length is longer than the
CT length.
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66
decreased. As the DNA length increases beyond the maximum
DNA CT length, the probability that two or more [4Fe4S]
cluster proteins bind to DNA far enough away from one
another to be outside of the range for DNA CT should
increase, and the proportion of unperturbed [4Fe4S]
3+
binding
should increase with it. With this in mind, we conducted
experiments using DNA of di
ff
erent lengths and measured the
binding of [4Fe4S] proteins to see if we could observe
di
ff
erences in the number of bound proteins per base pair and
thereby determine the maximum e
ff
ective length of DNA CT
between two proteins. Within the context of our proposed
model, the e
ff
ective distance is de
fi
ned as the length of DNA
within which two proteins are able to in
fl
uence their ensemble
a
ffi
nity to the DNA duplex via CT. Notably, the e
ff
ective CT
length is not determined by directly measuring the length of
DNA in between the proteins given our inability to distinguish
proteins in di
ff
erent redox states. We instead monitor the
ensemble distribution of proteins on DNA after CT has
occurred.
RESULTS
DNA CT Length of WT EndoIII at Room Temperature
(RT).
The number of proteins bound to DNA was counted
using images collected using AFM techniques. The number of
proteins bound to di
ff
erent lengths of DNA was used to
determine the a
ffi
nity of those proteins to DNA. AFM images
were collected using double-stranded DNA of 1625, 1999,
2686, 3895, 5153, 5967, 7037, 7996, and 8922 bp in length
prepared using PCR and plasmid excision methods (
Scheme
1
). The AFM results are summarized in
Figure 2
and
Figure
S1
. Two regimes are observed, designated by black and blue
lines. The blue line, with a shallow slope, represents the regime
where the DNA length is shorter than the CT length. Here the
[4Fe4S] cluster proteins are within CT distance for all binding
con
fi
gurations. Increases in DNA length in this regime
e
ff
ectively add a lower a
ffi
nity binding site, giving it a relatively
shallow slope. The steeper black line represents the regime
where DNA length is longer than the CT length. Here some
[4Fe4S] cluster proteins are not within CT distance to
communicate with each other. As the DNA CT length
increases, the probability of [4Fe4S] cluster proteins not
within the CT distance with each other increases, giving them
a higher e
ff
ective a
ffi
nity and resulting in a steeper slope.
Although the average number of bound proteins is less than
one, the presence of DNA duplexes bound with 2 and 3
proteins shows that the binding of proteins to DNA of these
lengths is in
fl
uenced by DNA CT, and our data indicate that
the prevalence of DNA with 2 and 3 proteins is suppressed by
DNA CT. These data suggest that proteins in the 2+ and 3+
oxidation states within CT distance have a lower ensemble
binding a
ffi
nity than proteins that are isolated from each other.
The point where the two regimes converge is the DNA length
where it starts to become possible that proteins bind without
experiencing DNA CT; we consider this the value for the
maximum DNA CT length between proteins. At ambient
temperatures, WT EndoIII exhibits a DNA CT length of 3509
±
509 bp, or 1.192
±
0.174
μ
m(
Figure 2
and
Figure S1
),
signi
fi
cantly longer than shown by previous measurements.
DNA CT Length of Y82A EndoIII Mutant at RT.
To
determine whether the observed slope change is in fact related
to DNA CT, we performed control experiments with the Y82A
EndoIII mutant that was previously characterized to be DNA
CT-de
fi
cient with its enzymatic activity unperturbed.
16
The
AFM results are summarized in
Figure 3
with additional
information in
Figure S2
. Only one regime is observed with no
apparent change of slope as a function of DNA lengths. Since
Scheme 1. Constructing Linear Double-Stranded DNA of
Various Lengths by (a) Plasmid Excision Using SacI
(Magenta) and (b) PCR Ampli
fi
cation Methods
a
a
The detailed protocols to prepare DNA duplexes of di
ff
erent lengths
are presented in the
SI
, Methods section. See
Table S1
for the primer
sequences and plasmid templates used in the PCR ampli
fi
cation steps.
Figure 2.
Measuring DNA-binding density of wild-type EndoIII at
ambient temperature using atomic force microscopy. A sharp change
in binding slope is observed at 3509 base pairs, or about 1.2
μ
m, that
suggests the presence of two regimes for protein binding, one within
the range for DNA CT (regime 1, blue), one outside of the range for
CT (regime 2, black). 1310 DNA duplexes were analyzed from four
independent preparations.
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Y82A does not exhibit DNA CT but remains otherwise the
same as WT EndoIII,
61
the lack of the slope change in
Figure 3
is consistent with the slope change observed for WT EndoIII
in
Figure 2
being a result of DNA CT. As Y82A does not
undergo redox signaling with each other via DNA CT, the
DNA-binding a
ffi
nity of Y82A is independent of each other.
Y82A does not exhibit DNA CT but remains otherwise the
same as WT EndoIII.
12
,
61
Thus, these data in
Figure 3
show
that DNA CT de
fi
ciency is su
ffi
cient to eliminate the two-
sloped feature observed in data collected with the wild-type
protein in
Figure 2
, which suggests that the change in slope in
the wild-type protein is a result of DNA CT.
DNA CT Length of WT EndoIII at 4
°
C.
To examine if the
DNA CT length is temperature dependent, we conducted
AFM experiments with samples prepared at colder temper-
atures. The AFM results are presented in
Figure 4
.At4
°
C,
WT EndoIII exhibits a much longer DNA CT length (5383
±
174 bp, or 1.830
±
0.059
μ
m) than that measured at RT. This
increase in DNA CT length is expected, as a decrease in
temperature limits molecular motion; i.e., DNA will be less
fl
oppy.
62
DNA CT Length of WT EndoIII at RT in the Presence
of a Metallointercalator.
To determine if the DNA CT
length can be altered using molecular means, we conducted
AFM experiments in the presence of [Ru(phen)
2
dppz]Cl
2
(50
μ
M), which is a known metallointercalator.
63
The redox
potential of the [4Fe4S] clusters in EndoIII is outside of the
potential range at which the Ru intercalators are reduced or
oxidized.
64
The AFM results are displayed in
Figure 5
with
additional information in
Figure S3
. The AFM experiments
demonstrate that in the presence of 50
μ
M [Ru(phen)
2
dppz]-
Cl
2
WT EndoIII exhibits a DNA CT length of 5399
±
1732
Figure 3.
Measuring DNA-binding density of Y82A EndoIII mutant
at ambient temperature using atomic force microscopy. No change in
binding slope as a function of DNA lengths is observed, indicating the
absence of DNA CT. As Y82A does not undergo redox signaling with
each other via DNA CT, the DNA-binding a
ffi
nity of Y82A is
independent of each other. 272 DNA duplexes were analyzed from
four independent preparations.
Figure 4.
Measuring DNA-binding density of wild-type EndoIII at 4
°
C using atomic force microscopy. A sharp change in binding slope
occurs at 5383 base pairs, or about 1.8
μ
m, indicating that the DNA
CT length observed at 4
°
C is signi
fi
cantly longer than that at RT. 151
DNA duplexes were analyzed from four independent preparations.
Figure 5.
Measuring DNA-binding density of wild-type EndoIII in the
presence of [Ru(phen)
2
dppz]Cl
2
(50
μ
M) at ambient temperature
using atomic force microscopy. A sharp change in binding slope
occurs at 5399 base pairs, or about 1.8
μ
m, suggesting that
metallointercalators lengthen the DNA CT length observed. 449
DNA duplexes were analyzed from four independent preparations.
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68
bp, or 1.836
±
0.589
μ
m(
Figure 5
and
Figure S3
). This
increase in DNA CT length is consistent with the increase in
DNA stability upon intercalation of metal complexes.
63
,
65
,
66
Additionally, as one would expect, the results support the
presence of
π
-stacked metallointercalators not impeding DNA
CT.
DISCUSSION
Determining DNA CT Length Using AFM Experi-
ments.
This study aims to measure the e
ff
ective maximum
distance for DNA-mediated charge transport between proteins
by monitoring changes in the a
ffi
nity of [4Fe4S] proteins to
DNA with increasing duplex length. An obvious change in
slope occurs for EndoIII that is capable of DNA CT, but not
for its CT-de
fi
cient mutant Y82A. Although the average
number of proteins bound is less than one in some cases, these
data show binding of the second and higher number of
proteins is signi
fi
cantly decreased beyond what would be
expected if the second (and higher) protein bound
independently of the
fi
rst, which shows that this aggregate
value is taking into account the in
fl
uence of DNA CT on
binding a
ffi
nity. We can therefore consider the change in slope
a feature of proteins capable of DNA CT, which indicates that
an increase in a
ffi
nity with DNA length is shallow at shorter
DNA distances and steep at longer DNA distances. Addition-
ally, it is possible to modify the point at which this slope
change occurs by changing the temperature and including
DNA intercalators, both of which can change the stability of
the duplex and the e
ffi
ciency of DNA CT.
Understanding DNA CT Length Using an Equilibrium
Binding Model.
The data collected using AFM represent a
snapshot of the proteins bound to DNA at equilibrium. Fitting
a plot of the number of proteins per duplex from AFM data
shows an intersection of two di
ff
erent regimes, whereby
increasing the length of DNA di
ff
erently a
ff
ects the increase in
a
ffi
nity of proteins for the additional binding sites. We interpret
this change in slope as indicative of a maximum CT length at
ambient temperature of 3509
±
509 bp, or 1.192
±
0.174
μ
m,
which is not observed in CT-de
fi
cient proteins.
To further understand these data, an equilibrium model was
developed to help describe the DNA-binding behavior of
proteins that are capable of DNA CT (see the
SI
, Methods).
The
fi
rst protein that binds to DNA is insensitive to DNA CT,
and the a
ffi
nity we observe is dependent on the oxidation state
of the protein [4Fe4S] cluster.
38
The dissociation constant for
EndoIII with a reduced [4Fe4S]
2+
cluster was previously
determined by MST to be 1.3
×
10
4
per bp, and EndoIII with
an oxidized [4Fe4S]
3+
cluster has an a
ffi
nity of 2.3
×
10
7
per
bp.
38
These values were used as inputs of our model to
estimate the percent of oxidized [4Fe4S]
3+
clusters in our
sample, 5.6
±
1.7% (see the
SI
, Methods). Modeling the
binding of additional proteins to incorporate contributions of
DNA CT allows us then to determine the e
ff
ective dissociation
constant for proteins undergoing CT, which is 4.3
×
10
5
±
1
×
10
5
per bp, a decrease in a
ffi
nity of over 2 orders of
magnitude compared to the oxidized protein that results from
electron transfer with a nearby reduced protein.
Importantly, using these values for the percent of oxidized
[4Fe4S] clusters and the e
ff
ective a
ffi
nity of proteins
undergoing CT, we may simulate the data collected by AFM
(
Figure S4
). Results from our theoretical calculations show the
same trends as the collected data, including the two regimes
above and below the e
ff
ective CT distance with di
ff
erent
slopes. The intersection of these two regimes in the simulated
data provides a maximum CT length of 2990 bp, which agrees
well with the experimental data and further strengthens the
claim that the presence of two regimes in our experimental
data is due to the e
ff
ect of DNA CT on the DNA-binding
a
ffi
nity of redox-active proteins.
Other experimental conditions yield similar values for the
percent of oxidized protein and decrease in a
ffi
nity due to
charge transport but show dramatic di
ff
erences in the length of
CT (
Table S3
). Fitting similar data collected from a CT-
de
fi
cient Y82A EndoIII mutant does not show any measurable
CT distance, which is also supported by simulated data using
the same model (
Figure S4
). Decreasing the temperature at
which proteins are incubated with DNA to 4
°
C increases the
measured CT length to 5383
±
174 bp. As temperature
decreases, the persistence length of the DNA backbone
increases.
62
The incorporation of the metallointercalator
[Ru(phen)
2
dppz]Cl
2
,whichisknowntostabilizeDNA
duplexes,
63
,
65
,
66
increases the CT length to 5399
±
1732 bp.
These results suggest that as the rigidity of DNA increases, the
CT length also increases, likely due to stabilizing the DNA
duplex in a more CT-favorable state.
Biological Implications of DNA CT Length.
Knowing
the length that DNA CT can occur between proteins allows for
predictions to be made about the e
ff
ectiveness of DNA CT in
increasing the e
ffi
ciency of DNA damage repair. In our initial
prediction, by assuming a DNA CT length of 200
500 bp, the
time needed to search the
E. coli
genome decreases by an order
of magnitude over calculations excluding DNA CT.
16
The
current AFM study indicates a DNA CT length of 3500 bp, so
that the e
ffi
ciency of the genome lesion search can signi
fi
cantly
increase further or require fewer involved proteins. Incorporat-
ing the data from this AFM study will be useful for better
modeling of the DNA damage search and repair process and
the role that such long-distance CT may play.
Now we consider how DNA CT plays a role in biological
systems using
E. coli
strain as an example.
E. coli
K-12 strain has
a genome size of 4 639 221 bp.
67
Dividing the number of total
bp by the measured DNA CT length of 3509 bp per protein
gives a rough calculation that 1322 [4Fe4S] repair proteins are
needed to place the whole
E. coli
genome within DNA CT
range of these [4Fe4S] repair proteins. Although individually
these [4Fe4S] repair proteins may be present in cells in low
copy numbers, previous studies have demonstrated that
[4Fe4S] cluster proteins from di
ff
erent repair pathways can
signal with each other
in vivo
.
11
Summing up the copy numbers
of EndoIII, MutY, and DinG gives an estimate of the total
number of known [4Fe4S]-containing DNA repair proteins in
E. coli
of at least about 1100.
15
We have measured the redox
potential of multiple proteins, and
fi
nd that they are all close to
one another, which suggests that they can all in
fl
uence one
another
s binding a
ffi
nity through DNA CT. However, it is not
clear how other di
ff
erences within the cell may a
ff
ect the search
and repair process. With more [4Fe4S] proteins yet to be
described, together these relevant [4Fe4S] repair proteins may
participate in and enable a genome-wide search for DNA
lesions via DNA CT.
CONCLUSIONS
In this report, we utilized atomic force microscopy and an
equilibrium binding model to study the e
ff
ective DNA charge
transport length between oxidized and reduced Endonuclease
III, a DNA repair protein carrying a redox-active [4Fe4S]
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Research Article
DOI:
10.1021/acscentsci.8b00566
ACS Cent. Sci.
2019, 5, 65
72
69
cluster. AFM data reveal two regimes of DNA-binding
behavior. In one regime, where the DNA length is shorter
than the CT length, a copy of EndoIII in the reduced
[4Fe4S]
2+
state bound on DNA modulates the DNA-binding
a
ffi
nity of a second copy of EndoIII with an oxidized [4Fe4S]
3+
cluster, no matter where the second copy is bound on DNA.
Because the two EndoIII proteins are always within CT
distance, their e
ff
ective a
ffi
nity for DNA is averaged. In another
regime where the DNA length is longer than the CT length,
there now exist binding sites where proteins with oxidized
[4Fe4S]
3+
clusters can bind outside of CT range with a higher
e
ff
ective a
ffi
nity. As the DNA length increases, the probability
of additional EndoIII binding outside of CT distance with
higher e
ff
ective a
ffi
nity increases. Modeling allowed us to
extract the maximum e
ff
ective DNA CT length between
proteins, which we found to be 3509 base pairs, or about 1.2
μ
m at ambient temperature. A mechanistic understanding of
this long-range DNA CT now requires exploration by
theorists.
68
The DNA CT length can be changed with
temperature and by stabilizing DNA with metallointercalators.
These data suggest that the e
ff
ective DNA CT length can be
controlled by its environment, which has interesting
implications for both biological systems and technology that
use DNA CT. An e
ff
ective DNA CT length this long enables a
fast genome lesion search scheme via long-range redox
signaling.
EXPERIMENTAL SECTION
General Procedures.
Chemicals were obtained from
commercial sources (Sigma-Aldrich, Fisher Scienti
fi
c, VWR,
and New England Biolabs) and used without further
puri
fi
cation unless otherwise speci
fi
ed. DNA primer sequences
were purchased from Integrated DNA Technologies, puri
fi
ed
by high performance liquid chromatography (HPLC, HP 1100,
Agilent), characterized using matrix-assisted laser desorption
ionization (MALDI) mass spectrometry using an Auto
fl
ex
MALDI TOF/TOF instrument (Bruker), and quanti
fi
ed using
a 100 Bio UV
vis spectrophotometer (Cary, Agilent) as
described previously.
69
73
Phosphate bu
ff
er (pH 7.0, 5 mM
NaH
2
PO
4
, 50 mM NaCl) was prepared using Milli-Q water
(>18 M
Ω
cm). Experiments performed were replicated at least
three times using di
ff
erent samples, and data presented are
from representative trials. The
SI
, Methods section, contains
protocols for overexpression and puri
fi
cation of wild-type
EndoIII and Y82A mutant and synthesis of DNA duplexes of
various lengths (
Table S1
).
AFM Experiments.
AFM was conducted following
protocols reported previously.
11
,
38
,
60
,
61
Brie
fl
y, mica surfaces
were freshly cleaved with tape. Protein stock solution (100
nM) contained either WT EndoIII or Y82A mutant in
phosphate bu
ff
er (pH 7.0, 5 mM NaH
2
PO
4
, 50 mM NaCl).
Stock DNA solution (9
μ
M bp) contained the mixture of DNA
duplexes of various lengths in Tris elution bu
ff
er (EB, 10 mM
Tris-HCl, pH 8.5, Qiagen). A solution (23.5
μ
L) with a
fi
nal
protein concentration (12 nM) and a
fi
nal total DNA
concentration (5
μ
M bp) was prepared and incubated at
ambient temperature for 1 h or 4
°
C for 2 h to allow for the
loading of protein onto DNA to reach equilibrium while
minimizing cluster degradation. For experiments with metal-
lointercalators, a
fi
nal concentration of 50
μ
M[Ru-
(phen)
2
dppz]Cl
2
was added. MgCl
2
(200 mM, 1.5
μ
L per 25
μ
L total volume) was added to promote DNA adsorption on
mica for AFM experiments. After pipetting 12.5
μ
L of DNA/
protein/MgCl
2
solution onto a freshly cleaved mica surface
and incubating for 2 min, a continuous stream of Milli-Q water
(2 mL) was slowly poured over the top portion of the modi
fi
ed
mica surface while holding the piece of mica in a vertical
position to linearize the DNA. A piece of lint-free wipe was
used to dab dry the bottom edge of the mica surface. The
surface was dried using a stream of N
2
fl
owing in the same
direction as the water rinse for 2 min. No signi
fi
cant safety
hazards were encountered.
AFM Instrumentation.
FESPA-V2 AFM tapping mode
probes (Bruker Nano, Inc.) with a mean force constant of 2.8
N/m and a mean resonance frequency of 75 kHz were used in
a MFP-3D AFM instrument (Asylum Research). Images were
captured in air with scan areas of 5
×
5
μ
m
2
in tapping mode at
a scan rate of 1 Hz to obtain images of quality high enough for
AFM assay analysis (512 pixels/line, 512 lines/image). Over
1000 AFM images were collected, and over 2000 DNA strands
were analyzed blind as described previously.
38
AFM Image Analysis.
WSxM software (Igor Pro) was
used to analyze DNA contour lengths and height pro
fi
les of the
proteins as described previously.
38
DNA and proteins were
identi
fi
ed using the relative di
ff
erential height pro
fi
les between
protein and DNA. For each data set, images from at least three
independent samples were analyzed. Distinguishable DNA and
proteins were counted by hand. Duplexes that were over-
lapped, indistinguishable, or cut o
ff
by the edge of the image
were excluded from the counting procedure. Binding density is
de
fi
ned as the number of the proteins bound on a DNA
duplex. Data presented are from representative trials, and error
bars represent standard error of all trials based on the total
number of proteins observed (
Table S2
,
n
> 200 for all
experiments).
ASSOCIATED CONTENT
*
S
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website
at DOI:
10.1021/acscents-
ci.8b00566
.
Supplemental methods, data, and
fi
gures including
schematics, protein counts, box plots, UV
vis spectra,
and AFM image (
PDF
)
AUTHOR INFORMATION
Corresponding Author
*
E-mail:
jkbarton@caltech.edu
.
ORCID
Edmund C. M. Tse:
0000-0002-9313-1290
Jacqueline K. Barton:
0000-0001-9883-1600
Present Addresses
E.C.M.T.: Department of Chemistry, The University of Hong
Kong, Pokfulam Road, Hong Kong SAR.
T.J.Z.: Department of Chemistry and Chemical Biology,
Harvard University, Cambridge, MA 02138, USA.
Notes
The authors declare no competing
fi
nancial interest.
ACKNOWLEDGMENTS
We gratefully recognize the NIH (GM126904) for
fi
nancial
support. E.C.M.T. appreciates the Croucher Foundation for a
postdoctoral fellowship. T.J.Z. is an NSF fellow (DGE-
1144469). S.B. acknowledges Joseph L. Koo and Helen C.
ACS Central Science
Research Article
DOI:
10.1021/acscentsci.8b00566
ACS Cent. Sci.
2019, 5, 65
72
70
Koo for a student undergraduate research fellowship. We thank
Dr. Adam N. Boynton and Kelsey M. Boyle for providing
metallointercalators for this study. We are also grateful to the
Caltech Center for the Chemistry of Cellular Signaling for
instrumentation. This research was enabled from the use of the
Auto
fl
ex MALDI TOF in the Caltech CCE Multiuser Mass
Spectrometry Laboratory, acquired with funds from the DOW
corporation (CCEC.DOWINSTR). AFM experiments were
carried out at the Molecular Materials Research Center of the
Beckman Institute of the California Institute of Technology.
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