S
1
Supporting Information
ATP
-
stimulated DNA
-
mediated Redox Signaling by XPD, a DNA Repair and
Transcription Helicase
Timothy P. Mui
#
, Jill O. Fuss
†
,
Justin P. Ishida
†
,
John A. Tainer
†,
+
, and Jacqueline K. Barton
#,*
#
Division of Chem istry and Chem ical Engineering, California Institute of Technology, Pasadena
, California
91125
, United
States
†
Lif e S ciences Divisio n, Lawrence B erkeley Natio nal L
aboratory, Berkeley, California 94720, United States
+
Departm ent of M olec
ular Biology, Skaggs Institute of Chemical Biology, The Scripps R
esearch Institute, La Jolla, California
92037
, United States
Materials and Methods
Chemicals were purchased from Sigma Aldrich.
All reagents for DNA synthesis were
purchased
from Glen
Res
earch.
Gold on mi
ca was purchased from Agilent Technologies.
DNA Synthesis
DNA Sequences were synthesized using standard phosphoramidite chemistry at either
IDT DNA (Coralville, IA) or on an Applied Biosystems 3400 DNA synthesizer.
Thiol
additions
were introduced using disulfide phosphoramidites (Glen Research), which were subsequently
reduced prior to experiment. All oligonucleotides were purified by HPLC and confirmed by
mass
-
spec.
DNA Sequences
Supplementary
Table S1.
Representative
table of DNA duplexes utilized.
Duplex
Sequence
Duplex DNA
SH 5’
–
GTGCTGCAACGTGTCTGCGC
–
3’
3’
–
CACGACGTTGCACAGACGCG
–
5’
Overhang DNA
SH 5’
–
GTGCTGCAACGTGTCTGCGC
–
3’
3’
–
CACGACGTTGCACAGACGCGAGAGCAGAC
–
5’
Overhang DNA w/MM (high)
SH 5’
–
GT
GC
C
GCAACGTGTCTGCGC
–
3’
3’
–
CACGACGTTGCACAGACGCGAGAGCAGAC
–
5’
Reverse DNA
SH
5
’
–
CACGACGTTGCACAGACGCGAGAGCAGAC
–
3’
3’
–
GTGCTGCAACGTGTCTGCGC
–
5’
S
2
DNA Modfied Electrodes
DNA modified electrodes were prepared essentially as previously
described.
1
Briefly, a
50
μ
L solution of 50 mM duplex DNA (as determined by UV
-
Vis) was allowed to incubate on a
bare gold on mica surface at 4
°
C overnight. The free DNA was then removed and the surface
rinsed a minimum of three times with phosphate buffe
r (5 mM phosphate, 50 mM NaCl, pH 7.0).
Next 50
μ
L of a 1 mM 6
-
mercapto
-
1
-
hexanol solution in protein buffer was allowed to passivate
the surface at ambient temperature for 2 hours. Finally, the surface was rinsed a minimum of
three times with protein bu
ffer.
S
3
Protein Expression
The
Sulfolobus acidocaldarius
XPD expression construct as described
2
was modified to
improve expression by removing a frame
-
shifting ATG start codon between the RBS and the
correct start codon
. Mutants were then generated using Quikchange XL II kit (Stratagene) and
verified by sequencing. All proteins were expressed in BL21 Rosetta2
E. coli
cells (Invitrogen)
grown in Terrific Broth at 37ºC for 3 hours after induction with 0.2 mM IPTG. Cells
were
resuspended in 20 mM MES pH 6, 100 mM NaCl, 1 mM DTT, 1 mM EDTA and lysed by
sonication and constant cell disruptor (Constant Systems) at 20,000 psi. After centrifugation at
29,000 RCF for 30 min, the supernatant was heat treated (65ºC, 20 min), cen
trifuged as before
and incubated with Capto DEAE (GE Healthcare) for 10
-
20 min at room temperature. The flow
-
through was collected by gravity and loaded onto a 5 mL HiTrap Heparin (GE Healthcare)
column at 2 mL/min. Bound protein was washed (7 column vol
umes) with lysis buffer and
eluted with a linear gradient (7 column volumes) to 45% high salt buffer (20 mM MES pH 6, 1
M NaCl, 1 mM DTT, 1 mM EDTA). SaXPD
-
containing fractions (~36% high salt buffer) were
pooled, concentrated using Amicon Ultra, Ultracel
-
10k regenerated cellulose spin filters, and
fractionated on a HiLoad 16/60 Superdex200 (GE Healthcare) size exclusion column at 1
mL/min in 10.8% high salt buffer (200 mM NaCl final). SaXPD
-
containing fractions were
pooled, concentrated as above to 2
-
12
mg/mL by OD
280
, and frozen. All cell and protein
manipulations were carried out at 4
°
C unless otherwise noted, buffers were filtered and
degassed, and purification was carried out in one day when possible to limit oxygen exposure.
Prior to electrochemi
stry measurements, proteins were dialyzed against the protein buffer
(20 mM Phosphate, 100 mM NaCl, 1 mM EDTA, 5% glyercol, pH 7.5) to remove residual DTT.
The concentration of individual proteins were determined by UV
-
Vis using
ε
=17,000 M
-
1
at 410
S
4
nM for
the [4Fe
-
4S] cluster.
3
For a representative trace see
Supporting Information Figure S4
.
Most protein work was conducted in an oxygen
-
free environment to prevent degradation of the
[4Fe
-
4S] cluster.
S
5
Protein Electrochemistry
Electrochemistry was
performed as previously described
1
with modifications indicated.
Measurements were obtained in an oxygen
-
free atmosphere using a CH Instruments 620C
electrochemical analyzer. The working electrode was a Au (111) chip with a platinum wire
auxiliary. Ag/A
gCl (Basi Electrodes) electrode was modified with a 4% agarose gel tip and
served as the reference. The 3
-
electrode setup was placed in a pseudo
-
closed tip box filled with
degassed water to prevent evaporation of the protein from the electrode. Protein wa
s added in
aliquots of 40
-
50
μ
L with concentrations as indicated.
Both ATP and ATP
-
γ
-
S were purchased from Sigma and dissolved in protein storage
buffer to make 100 mM stock solutions that were subsequently degassed with argon. For ATP
and ATP
-
γ
-
S addition
s, the appropriate degassed stock solution was added and mixed by
pipetting.
For data analysis, current values were taken by averaging the reductive sweep of a CV
after linear base line corrections. After equilibration on the surface, a linear fit was u
sed as the
background current. The value of the current was then subtracted from the background current
to obtain the difference in current. This value was then normalized to the surface by dividing by
the background current to obtain the % difference in
current. From the % difference in current
data, 1
st
order kinetics were plotted to fit the parameters yield=1
-
e
-
kt
.
S
6
Biochemical Assays
Helicase assays were performed as described
2
with the following modifications. 100 nM
5’
-
FAM
-
labeled DNA substrate
and 50
-
500 nM XPD was used. Gels were vi
sualized and
quantified using a
VersaDoc MP 4000 Molecular Imager and Quantity One software,
respectively
(BioRad).
ATPase assays were carried out under the same conditions as the helicase assays except
that unlabe
led ssDNA (25
-
mer from helicase substrate) was used and the total reaction volume
was 50
μ
L. The amount of phosphate released was measured using Biomol Green Reagent (Enzo
Life Sciences) according to the manufacturer’s instructions in 96
-
well plates on an
Infinite
M1000 microplate reader (Tecan).
SaXPD
-
DNA binding interactions were measured by fluorescence anisotropy (FA) as
described
2
with the following modifications. 20 nM substrate was used, reactions were incubated
for 15 min, and FA was measured in
384
-
well plates using an Infinite M1000 microplate reader
(Tecan).
S
7
Supplementary
Figure
S
1
.
Electrochemist
ry of SaXPD on a DNA modified electrode
backfilled with mercaptohexanol (orange), on a mercaptohexanol only electrode (blue) and
without SaXPD
on a DNA modified electrode (red) (Ag/AgCl reference electrode; Pt auxiliary
electrode, 50 mV/s scan rate).
Th
ese data indicate
that this is a DNA
-
mediated process.
S
8
Supplementary
Figure
S
2
.
Cathodic sweep of CVs for
SaXPD [10
μ
M] on
DNA
modified
electrodes
pre and post [5 mM] ATP additio
n
(left). % Difference in current versus time for
SaXPD on DNA modified electrodes (right). The arrow
on the right
indicates addition of 5 mM
ATP.
Currents were obtained through averaging of the reducti
ve sweep of the
various
voltamm
o
grams
(Ag/AgCl reference electrode; Pt auxiliary electrode, 50 mV/s scan rate).
S
9
Supplementary Figure S3.
Plots as shown in Figure 2. Arrows have been included during the
ATP additions to indicate an initial phase as we
ll
as the evolution of the signal with ATP
hydrolysis.
S
10
Supplementary
Figure
S
4
.
UV
-
Vis spectrum of WT SaXPD. The shoulder at 410 nm, is
indicative of a [4Fe
-
4S] cluster and is used for determining the concentration of the XPD
mutants.
S
11
References
:
(1
)
Boal, A. K.; Yavin, E.; Lukianova, O. A.; O’Shea, V. L.; David, S. S.; Barton, J. K.
Biochemistry
2005
,
44
, 8397.
(
2
)
Fan, L.; Fuss, J. O.; Cheng, Q. J.; Arvai, A. S.; Hammel, M.; Roberts, V. A.; Cooper, P.
K.; Tainer, J. A.
Cell
2008
,
133
, 789.
(
3
)
Boal, A. K.; Genereux, J. C.; Sontz, P. A.; Gralnick, J. A.; Newman, D. K.; Barton, J. K.
Proc
.
Natl
.
Acad
.
Sci
. U.
S
.
A
.
2009
,
106
, 15237.