of 8
A Compass at Weak Magnetic Fields Using Thymine Dimer Repair
Theodore J. Zwang,
Edmund C. M. Tse,
Dongping Zhong,
*
,
and Jacqueline K. Barton
*
,
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States
Departments of Chemistry and Physics, The Ohio State University, Columbus, Ohio 43210, United States
*
S
Supporting Information
ABSTRACT:
How birds sense the variations in Earth
s magnetic
fi
eld for navigation is
poorly understood, although cryptochromes, proteins homologous to photolyases, have
been proposed to participate in this magnetic sensing. Here, in electrochemical studies with
an applied magnetic
fi
eld, we monitor the repair of cyclobutane pyrimidine dimer lesions in
duplex DNA by photolyase, mutants of photolyase, and a modi
fi
ed cryptochrome. We
fi
nd
that the yield of dimer repair is dependent on the strength and angle of the applied
magnetic
fi
eld even when using magnetic
fi
elds weaker than 1 gauss. This high sensitivity to
weak magnetic
fi
elds depends upon a fast radical pair reaction on the thymines leading to
repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be
used in a biological compass informed by variations in Earth
s magnetic
fi
eld.
INTRODUCTION
Migratory birds and other animals can detect Earth
s magnetic
fi
eld to guide navigation, though the mechanisms underlying
this magnetic sensing are unclear.
1
The two mechanisms
proposed to explain the phenomenon of avian magneto-
reception are not mutually exclusive and involve sensing using
(i) magnetically sensitive radical pairs or (ii) magnetic iron-
containing nanoparticles.
2
Photolyases are enzymes that repair
UV-induced lesions and contain a highly conserved core
structure that could be involved in such magnetosensitive
radical pair chemistry,
3
although experiments exploring the
magnetosensitivity of DNA-bound photolyase have not been
reported previously. The conserved region contains a redox-
active
fl
avin adenine dinucleotide (FAD) cofactor, which
absorbs blue light, and carries out electron transfers with
pyrimidine dimer lesions via a cavity in the center of a DNA-
binding groove.
4
,
5
We have developed electrochemical methods to monitor the
repair of cyclobutane pyrimidine dimer (CPD) lesions by
photolyase.
6
Escherichia coli
photolyase repairs a cyclobutane
pyrimidine dimer in a reductive catalytic cycle upon irradiation
of the fully reduced
fl
avin cofactor (FADH
) with blue light.
CPD lesions form as a result of a photoinduced [2 + 2]
cycloaddition between two adjacent pyrimidines, typically
thymines, on the same DNA strand and signi
fi
cantly kink
duplex DNA. We employ DNA-modi
fi
ed electrodes immersed
in aqueous bu
ff
er using DNA charge transport (DNA CT) to
monitor repair of the CPD lesion within a DNA oligonucleo-
tide duplex. DNA CT relies on charge moving
through
the
internal base pair stack of the DNA duplex, and the e
ffi
ciency of
DNA CT is extremely sensitive to disruptions in base stacking
such as those that arise with a CPD lesion.
7
Upon repair of the
CPD by photolyase, DNA regains its well-stacked structure and
is able to support e
ffi
cient DNA CT to the
fl
avin cofactor. As a
result, the repair of CPD lesions by photolyase is monitored as
an increase in electrochemical response, because the repair
directly improves the yield of DNA-mediated CT between the
electrode and
fl
avin.
Figure 1
illustrates this electrical
monitoring of repair through cyclic voltammetry (CV)
performed on one set of gold electrodes modi
fi
ed with duplex
DNA containing a CPD, bound by photolyase and irradiated
with blue light. When bound to the CPD, the redox-active
fl
avin is apparent at
120 mV versus AgCl/Ag, though the
signal is small, owing to the presence of the intervening CPD;
upon irradiation, the photolyase repairs the CPD and the signal
increases.
We can explore directly how a magnetic
fi
eld a
ff
ects the
DNA repair reaction carried out by photolyase using this
electrochemistry in the presence of a magnetic
fi
eld. Indeed, the
electrode serves to orient the DNA and DNA reaction relative
to the magnetic
fi
eld. Previous experiments have monitored
changes in the transient absorption spectra of photolyase in the
presence of a magnetic
fi
eld,
8
but in the absence of DNA; it is
therefore unclear from these experiments how photolyase
activity on its DNA substrate is a
ff
ected by a magnetic
fi
eld.
Indeed, we
fi
nd a remarkable sensitivity to low strength
magnetic
fi
elds in this reaction to repair cyclopyrimidine dimers
and not only by photolyase but also by a truncated
cryptochrome, the protein family thought to be responsible
for magnetoreception generally.
Received:
January 4, 2018
Published:
March 7, 2018
Research Article
Cite This:
ACS Cent. Sci.
2018, 4, 405
412
© 2018 American Chemical Society
405
DOI:
10.1021/acscentsci.8b00008
ACS Cent. Sci.
2018, 4, 405
412
This is an open access article published under an ACS AuthorChoice License, which permits
copying and redistribution of the article or any adaptations for non-commercial purposes.
RESULTS AND DISCUSSION
E
ff
ects of Magnetic Fields on the Photolyase
Reaction.
Multiplexed chips consisting of 16 separate DNA-
modi
fi
ed gold electrodes allow for the simultaneous or
sequential comparison of four distinct monolayers created
under identical conditions with 4-fold redundancy,
9
and thus
these multiplexed chips allow the evaluation of the e
ff
ects of
magnetic
fi
elds on the photolyase reaction in a well-controlled
system.
Figure 2
shows representative data from a single
multiplexed chip where two quadrants were incubated with the
same thiolated duplex DNA containing a thymine dimer
(T
T); the third quadrant contains duplex DNA with a C:A
mismatch intervening between the T
T and the gold surface,
and the last quadrant contains duplex DNA with no dimer or
mismatches. When photolyase is added to a monolayer of
duplex DNA, containing a T
T (29 bp duplexes,
8 pmol/
cm
2
) in the absence of an applied magnetic
fi
eld, irradiation
with blue light (405
±
10 nm) leads to the increase in current
for the FADH
redox couple. Importantly, the gold surface
uniformly orients the DNA as well as the DNA-bound
photolyase. Shining light on an identical monolayer but in
the presence of an applied magnetic
fi
eld, however, leads to a
signi
fi
cant decrease in the yield of charge transferred over the
same period of time. The lack of signal on the electrode
modi
fi
ed with DNA but without the T
T shows that the
photolyase binds speci
fi
cally to its substrate CPD lesion.
Furthermore, incorporating a single mismatch signi
fi
cantly
decreases the yield of charge transferred to the
fl
avin, indicating
that the
fl
avin is reduced and oxidized by charge transferred
through
the DNA duplex; perturbations to the base stack as
occurs with a mismatch are su
ffi
cient to decrease DNA CT.
Control experiments show that the protein is still active after
multiple hours of incubation in a magnetic
fi
eld and that the
protein binds competitively to CPD-containing duplex DNA
(
Figure S1
). There is also no distinguishable di
ff
erence when
adding this CPD-containing duplex DNA in the presence or
absence of a magnetic
fi
eld, suggesting that the magnetic
fi
eld
does not cause a signi
fi
cant change in photolyase a
ffi
nity for
CPD. Assays with a SQUID magnetometer furthermore show
that there is no magnetite on the electrode surface that is
in
fl
uencing this chemistry (
Figure S2
).
The magnetic
fi
eld in
fl
uence on the yield of DNA CT
depends upon when the magnetic
fi
eld is applied during the
reaction. The presence or absence of an externally applied
magnetic
fi
eld during
fl
avin photoreduction, before the
incubation of the protein with the duplex DNA substrate,
does not in
fl
uence the signal during repair (
Figure S3
).
Importantly, removing the magnetic
fi
eld during repair restores
the yield of charge transfer (
Figure 3
). After repair has been
completed, adding a magnetic
fi
eld has no in
fl
uence on the
yield of charge transferred. Randles
Sevcik analysis (
Figure S4
)
demonstrates that photolyase di
ff
uses away from surface of the
DNA-modi
fi
ed electrode when there is no applied magnetic
Figure 1.
Cyclic voltammetry of thymine dimer repair by photolyase. (Top) Reductive catalytic cycle of the
fl
avin cofactor in photolyase to repair
thymine dimers. (Bottom) Cyclic voltammetry on multiplexed chip electrodes modi
fi
ed with 29 bp dsDNA and back
fi
lled with mercaptohexanol.
The reaction cartoon on the electrode is shown above with corresponding CV below. (Left) Monolayer of duplex DNA (29 bp), each with a single
thymine dimer (red T
T), is scanned anaerobically at 100 mV/s in Tris bu
ff
er (50 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, pH 7.5).
(Center) Addition of
E. coli
photolyase (50
μ
M) shows a small
fl
avin redox peak centered around
100 mV vs AgCl/Ag, which is consistent with the
fully reduced
fl
avin. (Right) Irradiation with blue light repairs the thymine dimer over time and increases the yield of charge transferred through the
DNA duplex to and from the
fl
avin. After subtracting the background current (dotted line), the area under the reductive peak can be integrated to
give the total charge transferred to the
fl
avin.
ACS Central Science
Research Article
DOI:
10.1021/acscentsci.8b00008
ACS Cent. Sci.
2018, 4, 405
412
406
fi
eld, associated with repair of the CPD and lowering of the
protein a
ffi
nity, but the photolyase stays bound to the surface
with an applied magnetic
fi
eld. Together, these data indicate
that the presence of the magnetic
fi
eld during repair directly
inhibits the e
ffi
ciency of repair.
Figure 4
illustrates how the repair e
ffi
ciency varies with
magnetic
fi
eld strength and angle. Signi
fi
cantly, at weak
magnetic
fi
elds, the magnetic
fi
eld strength plays an important
role in the e
ffi
ciency of dimer repair. The background magnetic
fi
eld during our experiments was measured to be 0.4 G and
resulted in the highest yield of repair. However, applying an
additional magnetic
fi
eld perpendicular to the surface as weak
as 0.2 G results in diminished yield. Increasing the
fi
eld strength
further decreases the yield, but eventually the e
ff
ect is saturated;
applied
fi
elds of 30 and 6000 G result in the same magnitude
decrease in yield. As a control, we also examined the enzymatic
restriction of an oligonucleotide by
Hin
cII; as expected, this
reaction is not in
fl
uenced by the presence of a magnetic
fi
eld,
nor by irradiation (
Figure S14
).
Moreover, the angle of the magnetic
fi
eld relative to the
plane of the electrode signi
fi
cantly in
fl
uences the yield. A
magnetic
fi
eld perpendicular to the plane of the electrode
exhibits the largest e
ff
ect. Changing the angle of inclination to
45
°
diminishes the e
ff
ect, as does applying a
fi
eld parallel to the
plane of the surface. Interestingly, there is no di
ff
erence in yield
observed for a magnetic
fi
eld pointing perpendicularly up
versus perpendicularly down (
Figure S5
), which suggests that
only the angle of the
fi
eld and not the polarity direction of the
fi
eld is important.
10
These results clearly illustrate that the CPD reaction is
sensitive to low magnetic
fi
eld strengths and
fi
eld direction.
These data are reminiscent of experiments carried out by N. J.
Turro, who established conditions critical for observation of
reactions controlled by weak magnetic
fi
elds.
11
What is required
is a competition between two processes: one that is magnetic
fi
eld dependent and one that is magnetic
fi
eld independent.
Figure 5
illustrates the CPD repair reaction carried out by
photolyase.
12
Here radical pair formation followed by electron
transfer leads either to separation of the two repaired thymines
or to futile back electron transfer without repair. Thus, the
magnetic
fi
eld dependent radical pair a
ff
ects the e
ffi
ciency of
the subsequent bond-breaking repair reaction.
Mutations in Photolyase and the CPD Lesion.
To
examine the factors governing this reaction in more detail, we
tested mutants of photolyase that perturb the internal electron
transfer pathways. In particular, we would expect mutations that
a
ff
ect the lifetime of the CPD radical pair to be most sensitive
to magnetic
fi
eld e
ff
ects. Radical pairs that involve both the
Figure 2.
Integrated cyclic voltam
metry measurement of a
representative multiplexed chip over time irradiated. (Top)
Representation of the multiplexed chip and the di
ff
erent duplex
DNA monolayers and experimental conditions that were tested.
(Bottom) Plot of the area under the reductive peak, which gives the
total amount of charge transferred to the
fl
avin, over time irradiated.
The color of traces corresponds to the quadrants shown in the
representation above. In each case, 50
μ
M photolyase was added and
irradiated with blue light at
t
= 0 anaerobically in Tris bu
ff
er (50 mM
Tris-HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, pH 7.5). In the
green quadrant, the 29 bp dsDNA contained no thymine dimer. In the
red quadrant, the 29 bp dsDNA contained a thymine dimer and a C:A
mismatch between the dimer and the electrode surface. In the black
quadrant, the 29 bp dsDNA contained a thymine dimer. In the blue
quadrant, the same 29 bp dsDNA containing a thymine dimer was
used as was tested in the black quadrant, but the entire experiment was
conducted with a 560 G magnetic
fi
eld pointing perpendicularly up
intersecting the plane of the electrode. Standard error was plotted with
n
=4.
Figure 3.
Total amount of charge transferred over time irradiated with
varying magnetic
fi
eld conditions. First, 50
μ
M photolyase was added
to a monolayer of 29 bp dsDNA with T
T and irradiated with blue
light (
t
= 0) anaerobically in the absence (black) or presence (blue) of
a 30 G magnetic
fi
eld applied perpendicularly up intersecting the plane
of the electrode. At the time indicated by the dotted line the magnetic
fi
eld was either applied (gray) or removed (light blue) to switch the
magnetic
fi
eld conditions in a given experiment. Standard error was
plotted with
n
=4.
ACS Central Science
Research Article
DOI:
10.1021/acscentsci.8b00008
ACS Cent. Sci.
2018, 4, 405
412
407
fl
avin and dimer radical anion could serve also as magnetically
sensitive intermediates in the repair reaction. Photoactivation of
FAD initiates electron transfers along a conserved triad of
tryptophan residues that gives a
fl
avin radical (FAD
) and a
tryptophan radical (TrpH
+
) that have been shown through
transient absorption spectroscopy to be sensitive to weak
Figure 4.
Total amount of charge transferred over time irradiated with varying magnetic
fi
eld strengths and angles. In all experiments photolyase (50
μ
M) was added to 29 bp dsDNA-modi
fi
ed electrodes containing T
T and then irradiated with blue light (
t
= 0) anaerobically in Tris bu
ff
er (50 mM
Tris-HCl, 50 mM KCl, 1 mM EDTA, 10% glycerol, pH 7.5). (Top left) The background magnetic
fi
eld was 0.4 G, and the applied
fi
eld was added to
give the total
fi
eld strength listed to the right of the plot. (Top right) The magnetic
fi
eld was applied perpendicularly up intersecting the plane of the
electrode surface. The magnetic
fi
eld angle was varied by applying a 30 G
fi
eld (top right) or 0.4 G
fi
eld (bottom right) at either a 0
°
,45
°
,or90
°
angle relative to the plane of the electrode surface. (Bottom left) The approximate angles at which the magnetic
fi
elds intersect thymine dimers are
illustrated. The redox potential of the
fl
avin lies negative of the potential of zero charge of the working electrode. At this potential the duplexes line
up approximately normal to the electrode surface, meaning that the thymines are approximately parallel to the surface.
9
The largest magnetic
fi
eld
e
ff
ect occurs when the
fi
eld intersects the dimer perpendicular to the plane of the bases, and the weakest e
ff
ect occurs when the
fi
eld is parallel to the
plane of the bases. Standard error was plotted with
n
4.
Figure 5.
Radical repair scheme for cyclobutane pyrimidine dimers (CPD) based on previous work.
11
Forward electron transfer from the fully
reduced
fl
avin results in a radical residing on the CPD. First the C5
C5
bond splits, followed by either C6
C6
bond splitting or futile back
electron transfer to the CPD state. Following bond splitting, either the radical residing on the pyrimidine can undergo electron return to the
fl
avin,
resulting in the completion of the repair process, or the radical can facilitate CPD formation and undergo futile back electron transfer.
ACS Central Science
Research Article
DOI:
10.1021/acscentsci.8b00008
ACS Cent. Sci.
2018, 4, 405
412
408