www.sciencemag.org/content/355/6327/aag1789/suppl/DC1
Supplementary Materials for
The [4Fe4S] cluster of human DNA primase functions as a redox switch using
DNA charge transport
Elizabeth O’Brien, Marilyn E. Holt, Matthew K. Thompson, Lauren E. Salay, Aaron C. Ehlinger,
Walter J. Chazin,* Jacqueline K. Barton*
*Corresponding author. Email: [email protected] (J.K.B.); [email protected] (W.J.C.)
Published 24 February 2017, Science 355, aag1789 (2017)
DOI: 10.1126/science.aag1789
This PDF file includes:
Figs. S1 to S17
Tables S1 and S2
References
Fig. S1. DNA binding for WT and mutant p58C. WT and mutant p58C domains have similar DNA
binding affinities with low micromolar dissociation constants. Affinities were measured in aerobic
conditions by fluorescence anisotropy of FITC-labeled DNA substrates in 20 mM MES, pH 6.5, 50
mM NaCl. Background was subtracted from anisotropy values prior to plotting. KD values are mean
± SD over n=3 measurements for each variant.
Fig. S2. Cyclic voltammetry (A) and Square Wave Voltammetry (B) signals of p58C bound to
of well-matched (WM) DNA or DNA containing an abasic site (AB DNA) in the duplex
segment, in the presence and absence of an NTP pool. The p58C domain displays a reversible
redox signal centered at 140-150mV vs. NHE in the presence of ss/dsDNA and 500μM [ATP].
This suggests that the [4Fe4S] cluster in DNA primase is capable of undergoing reversible redox
activity when the enzyme is in its active form, bound to DNA and NTPs. All electrochemistry
was performed in anaerobic conditions with a Ag/AgCl reference, on 10μM [p58C] in 20mM
Tris, pH 7.2, 75mM NaCl. Cyclic voltammetry scan rate: 100mV/s, square wave voltammetry
scan frequency: 15 Hz.
Fig. S4. P58C does not produce a redox signal on ss/dsDNA in the absence of
electrochemical alteration. CV performed in anaerobic conditions on 16μM p58C using a
Ag/AgCl reference electrode, in 20mM Tris buffer, pH 7.2, 75mM NaCl. CV scans were
conducted at a 100mV/s scan rate.
Fig. S5. A) Iterative oxidation reactions on a single DNA-modified multiplex electrode surface.
Oxidation reactions of p58C indicate that charge passes, converting a reduced species to an oxidized
species at an applied potential of +412mV vs. NHE. This shows that after reduction in CV, p58C
can be oxidized again on a DNA-modified surface. B) Charge transfer (nC) in the cathodic peak
signal for each CV scan of the iteratively oxidized p58C sample. Charge transfer increases during
iterative oxidations, due to more p58C available at the DNA/solution interface during later trials.
C) Plot of CV charge transfer for iterative oxidation steps. Charge transfer in CV peak increases
over iterative scans, suggesting that electrochemical oxidation brings more p58C to the ss/dsDNA
substrate. Electrochemistry performed on 16μM p58C in 20mM Tris, pH 7.2, 75mM NaCl. CV
scans performed at 100mV/s scan rate.
A
B
Y309
Y345
Y347
Fig. S6. Comparison of p58C structures obtained with two different crystallization conditions.
A) Superposition of the two p58C structures, 5F0Q (red) and 3LQ9 (blue). B) Expanded region
of the 5F0Q structure showing the distribution of the three conserved tyrosine residues that
make up the proposed charge transport pathway.
Fig. S8. Structural comparison of WT and mutant p58C. A)Superposition of the WT p58C
(PDB 3L9Q) and Y347F p58C (PDB 5DQO) structures. B) Superposition of the WT p58C
(PDB 3L9Q) and Y345F p58C (PDB 517M) structures. In both panels, WT p58C is colored
blue and the tyrosine to phenylalanine mutant is red.
Fig. S9. CV scans of p58C Y345F. A) Electrochemically unaltered p58C tyrosine mutants display
no electrochemical signal on DNA. B) Electrochemically oxidized p58C tyrosine mutants display a
cathodic peak between -150mV and -160mV vs. NHE after oxidation at an applied potential of
+412mV vs. NHE; the peak signal is smaller than that observed for wild type. C) Tyrosine mutants
of p58C display no redox signal on DNA after electrochemical reduction at an applied potential of 188mV vs. NHE. All scans performed on 16μM p58C variant, in 20mM Tris, pH 7.2, 75mM NaCl,
at a 100mV/s scan rate. P58C Y345F is shown as a representative example of the mutants.
Fig. S10. Iterative Oxidations, followed by CV scans, of p58C Y345F on a single electrode
surface. A) Three bulk electrolysis reactions at an applied potential of +412mV vs. NHE
were performed in immediate succession on a single electrode surface. B) CV scans after
each successive oxidation. Charge transfer in the cathodic peak was 4.1nC, 5.3nC, and
6.3nC during oxidations 1, 2, and 3, respectively. The mutant p58C thus shows the same
general trend as WT but transfers less charge in bulk electrolysis and subsequent CV on the
DNA-modified surface. All scans performed on 16μM p58C variant, in 20mM Tris, pH 7.2,
75mM NaCl, at a 100mV/s scan rate. P58C Y345F is shown as a representative example of
the mutants.
Figure S11. Change in potential value for cathodic peak after p58C oxidation, WT and tyrosine
mutants of p58C. Potential measurements from three CV trials after p58C electrochemical
oxidation show that the WT potential is more negative on average, but the difference between WT
and mutant p58C cannot be determined to a significant degree outside the margin of error. This
suggests that the mutants are more likely to be in the reduced [4Fe4S]2+ state than the WT protein
with an intact charge transfer pathway, as expected. Potential values are reported as mean ± SD
over n= 5 trials.
0
WT p58C
-0.05
Ered (V vs. NHE)
p58C Y345L
-0.1
-0.15
-0.2
-0.25
-0.3
-1.2
-1
-0.8
-0.6
-0.4
log (Scan Rate)
-0.2
0
Figure S12. Scan Rate dependence of cathodic peak potential in p58C CV signal after
electrochemical oxidation. The peak potential varies with the log of the scan rate for WT p58C
(blue points, solid line) and for p58C Y345L (green points, dashed line), as is expected for an
irreversible signal (55) in CV. The similar linear relationships of the potential and log of the
scan rate for these two relationships demonstrate that the rate-determining step in generating
this CV signal is tunneling through the alkanethiol linker tethering the DNA substrate to the
gold electrode surface.
Fig. S13. DNA binding for WT and mutant primase. WT and mutant primase have similar DNA
binding affinities with high nanomolar dissociation constants. Affinities were measured by
fluorescence anisotropy of FITC-labeled DNA substrates in the presence of atmospheric oxygen in 20
mM HEPES, pH 7.5, and 75 mM NaCl. Background was subtracted from anisotropy values prior to
plotting. KD values are mean ± SD over n=3 measurements for each variant.
Fully Elongated
(60 nt) products,
no truncation
Truncated
Exogenous
Primer Products
Figure S14. Gel separation of products for elongation reactions with increasing concentrations of
p48/p58 and p48/p58Y345F on 2’-OMe RNA-primed DNA. WT primase, which has an efficient redox
switching pathway between bound DNA and the [4Fe4S] cluster, synthesizes slightly more truncated
products on average. The CT pathway through the protein is important for the redox switch, but it does
not appear to be the sole mediator of primase product truncation. Elongation assays were performed
anaerobically, with 500nM primed DNA, 1 μM α-32P ATP, 120 μM CTP, 180 μM UTP, 200-800 nM
enzyme in 50 mM Tris, pH 8.0, 3 mM MgCl2, at 37 °C.
Fig. S15. Percent of primase products truncated by WT and CT-deficient variants at 5, 10, and 30
minutes of incubation at 37°C, under anaerobic conditions. Primase mutants are identified by the
mutation in the p58C domain. WT and mutant primase elongation activity at t= 5 minutes (top left), t=
10 minutes (top right), and t= 30 minutes (bottom left) of incubation. Legend is shown on the bottom
right. All measurements are mean ± SD for n= 3 trials.
Fig S16. Quantification of 60-nt products synthesized by WT and CT-deficient variants at 5,
10, and 30 minutes of incubation at 37°C, under anaerobic conditions. The products,
quantified by calculating 32P counts/minute from α-32P ATP incorporated into products
separated by mass on a 20% PAGE denaturing gel, are identical between variants within error
for each time point. The 60-nt products were quantified and compared to assess the nucleotide
polymerization of each variant, as these products are independent of regulation by the redox
switch. This demonstrates that CT-deficient primase retains the ability to polymerize
nucleotides and elongate exogenous primers. Conditions are shown for 400nM enzyme
concentration reactions. All measurements are mean ± SD for n= 3 trials.
**
##
***
***
**
**
#
***
Figure S17. Percentage of WT primase truncated products for a) t = 30 minutes (left) and b) t = 120
minutes (right). Similar to the 60 minute time point (incubation at 37°C), these plots suggest that CTproficient primase synthesizes significantly more truncated products on well-matched, primed DNA than
in the presence of a single base mismatch within the RNA primer. When primase cannot participate in
DNA CT, it favors product elongation to primer-multimer length, at all concentrations of CT-proficient
primase assayed. Mean ±SD values are plotted for n= 3 trials, # = 0.025<p<0.01, ## = 0.005<p<0.01, **
= 0.001<p<0.0005, *** = p<0.0005 (student’s t-test).
Table S1. Electrochemistry, primase activity assay, and fluorescence anisotropy DNA substrates used.
Electrochemistry of p58C was performed on self-assembling monolayers of a 20-mer DNA duplex
substrate with a 3-nt 5’- ssDNA overhang. A 50-nt ssDNA substrate with a single thymine base
complementary to the α-32P radiolabeled ATP was used in the primase initiation assay comparing wild
type and CT-deficient full-length enzyme. A 2’-OMe RNA-primed ss/dsDNA substrate, containing a
31- nucleotide duplex segment and a 29- nucleotide 5’-ssDNA overhang was used to assay elongation.
The mismatched elongation assay was performed with a cytosine engineered into the substrate (red) to
promote a mismatch in the elongated primer segment. U = 2’-OMe rU, SH = -(CH2)6-SH, Flc = FITC
Table S2
Crystallographic data collection and refinement statistics.
PDB Entry
517M
5DQO
Protein Structure
Y345F
Y347F
C2
P31
109.9, 52.7, 89.2
90, 115.25, 90
100
0.987
49.7-1.93
(1.96-1.93)
60.40, 60.40, 246.73
90, 90, 120
100
0.999
44.14- 2.48
(2.53-2.48)
142, 467
35, 604
99.9 (99.8)
184, 871
41, 462
99.1 (89.6)
Data Collection
Space Group
Cell dimensions
a, b, c (Å)
α, β, γ (°)
Temperature (K)
Wavelength (Å)
a
Resolution (Å)
Reflections
Total
Unique
a
Completeness (%)
Rmerge (%)
Rpim (%)
a,b
a
a
CC1/2 (%)
I/σI
Redundancy
8.7 (58.4)
5.8 (49.3)
0.048 (0.33)
2.9 (33.9)
(88.6)
18.7 (2.1)
4.2 (4.0)
(59.2)
27.6 (2.1)
4.5 (2.9)
21.0/24.9
24.9/27.4
314
2
81
628
4
41
28.8
21.5
27.7
24.5
0.02
2.11
53.69
46.16
55.66
58.1
0.02
1.91
99%
1%
98.5%
1.5%
0%
0%
Refinement
c
Rwork/Rfree (%)
No. of residues
Protein
4Fe-4S
Solvent
Average B-Factor (Å)
Protein
4Fe-4S
Solvent
2
Wilson B-Factor (Å )
RMSD Bonds (Å)
RMSD Angles (°)
Ramachandran (%)
Most favored
Allowed
Disallowed
a
Values in parentheses are for the highest-resolution shell. bRmerge = Σ(|I-Ī|)/Σ I x 100. cRwork =
Σ |F0-Fc|/ΣF0 x 100, where F0 is the observed structure factor amplitude and Fc is the
calculated structure factor amplitude.
Table S2. Crystallographic Data for p58C Y345F (PDB 517M) and p58C Y347F (PDB
5DQO).
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