of 5
Response to Comments on “The [4Fe4S] cluster of human DNA
primase functions as a redox switch using DNA charge
transport”
Elizabeth O’Brien
1
,
Marilyn E. Holt
2
,
Matthew K. Thompson
2
,
Lauren E. Salay
2
,
Aaron C.
Ehlinger
2
,
Walter J. Chazin
2,*
, and
Jacqueline K. Barton
1,*
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
CA 91125, USA
2
Departments of Biochemistry and Chemistry, Center for Structural Biology, Vanderbilt University,
Nashville, TN 37235, USA
Abstract
Baranovskiy
et al.
and Pellegrini argue that, based on structural data, the path for charge transfer
through the [4Fe4S] domain of primase is not feasible. Our manuscript presents electrochemical
data directly showing charge transport through DNA to the [4Fe4S] cluster of a primase p58C
construct and a reversible switch in the DNA-bound signal with oxidation/reduction, which is
inhibited by mutation of three tyrosine residues. Although the dispositions of tyrosines differ in
different constructs, all are within range for microsecond electron transfer.
Our study (
1
) provides electrochemical and biochemical data for human DNA primase,
supporting a proposal that [4Fe4S] cluster redox chemistry and DNA charge transport are
elements in the binding and termination of the polymerase activity of the primase. We
describe (i) electrochemical experiments on DNA-modified electrodes demonstrating that
the p58C domain of DNA primase exhibits tighter binding with the [4Fe4S] cluster in the 3+
state compared with the 2+ state; (ii) mutagenesis experiments showing that when any of
three tyrosine residues are mutated to phenylalanine, the ability to carry out the redox
reaction on the cluster electrochemically is inhibited and also that charge transfer–deficient
mutants exhibit reduced ability to initiate priming; and (iii) primer elongation and
termination assays with native full-length primase showing that the introduction of a
mismatch into the growing primer inhibits termination, which is consistent with the
regulation of primase termination by mismatch-sensitive DNA-mediated charge transport.
These data support our proposal that electron transfer between clusters in primase and
polymerase
α
, mediated by the growing DNA/RNA duplex, contributes to handoff of the
substrate between the two enzymes.
We emphasize that all electrochemistry and corresponding control experiments were
performed on the truncated p58C construct, which contains the [4Fe4S] cluster, and all
activity assays and corresponding control experiments were performed on the full-length
*
Corresponding author. jkbarton@caltech.edu (J.K.B.); walter.j.chazin@vanderbilt.edu (W.J.C.).
HHS Public Access
Author manuscript
Science
. Author manuscript; available in PMC 2018 May 04.
Published in final edited form as:
Science
. 2017 July 21; 357(6348): . doi:10.1126/science.aan2762.
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p48/p58 enzyme. Neither Baranovskiy
et al.
(
2
) nor Pellegrini (
3
) directly dispute the
electrochemical observations of charge transport through the DNA substrate to the iron-
sulfur cluster of our p58 construct, nor do they dispute its inhibition by mutation of each of
the three relevant tyrosine residues. Rather, they question the path for electron transfer
through primase on the basis of differences in structures of p58C, the primase domain
containing the [4Fe4S] cluster (
4
6
). The structure of human p58C (
4
) from which we
identify conserved tyrosine residues participating in a charge transfer pathway contains
residues 318 to 353 folded in a
β
-hairpin arrangement. Other structures of human (
5
) and
yeast (
6
) p58C show these residues in an
α
-helical arrangement and show that conserved
tyrosines (Y309, Y345, and Y347 for the human protein) in the charge transport pathway
through the protein are spaced and oriented differently in different structures. However, both
Comments do not appropriately consider the requirements for charge transport mediated by
tyrosines in a protein. Charge transfer through protein, which is only weakly dependent on
orientation, can occur over distances up to 15 Å on our proposed time scale and is most
accurately estimated from the distances between tyrosine centroids (
7
). In fact, regardless of
the differences in structures, the tyrosine centroids in all human and yeast p58C structures
are within feasible range for microsecond electron transfer through the protein matrix.
In addition to differences in crystallization conditions, our human p58C construct differs in
sequence from that of Baranovskiy
et al.
; our p58C construct corresponds to residues 272 to
464 from the human protein. Three additional residues on the N terminus of our construct—
essentially positions 269, 270, and 271—are left from the His tag used to purify the protein.
Tahirov uses a different truncation, including more residues on the N terminus but fewer on
the C terminus (266 to 456). Our p58C construct was used for the electrochemistry and
crystallography, but for all biochemical assays, we used the full human primase (no
mutations).
The Comments also raise concerns over our interpretation of data obtained for a Y345F
primase mutant, which results in reduced electron transfer efficiency. The argument is based
primarily on the assignment of a hydrogen bond between Y345 and the triphosphate group
at the 5
end of the DNA/RNA substrate in a crystal structure (
8
). We find it puzzling that
elimination of only one of more than 15 hydrogen bonds between p58C and the substrate is
proposed as an explanation for the dramatic change in biochemical activity that we observe.
In fact, binding assays in our laboratory comparing the full, wild-type primase enzyme and
the Y345F primase variant using the RNA/DNA substrate similar to the one used in the
crystal structure (
8
) show no detectable differences, as expected. Furthermore, the placement
of the Y345 side chain in the structure, which lies at the center of their claim, may be more
ambiguous than previously implied (
8
). Retrieving the coordinate files and electron density
maps for the structures (5F0Q), we observe a very poor fit for this residue. High B factors
for this residue relative to most other residues call into question whether the assigned
hydrogen bond has high enough occupancy to substantiate the argument. Moreover, we have
now carried out parallel biochemical experiments comparing primer synthesis of wild-type
full primase and the charge transfer–deficient Y309F variant of full p48/p58 primase on
single-stranded DNA (Fig. 1). Here, too, we observe significant inhibition of initiation in the
mutant, just as we observed with the Y345F and Y345C primase variants. Thus, mutation of
another tyrosine in the charge transfer pathway, one not interacting with any substrates, also
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inhibits initiation. Hence, inhibition of initiation is the result of inhibition of the redox
switch.
Baranovskiy
et al.
criticize our choice of substrate for the electrochemical studies of p58C.
Primase interacts with a range of DNA structures (
4
), enabling us to create a substrate that
productively binds p58C and satisfies the technical criteria needed for DNA-mediated
electrochemistry. Our substrates were designed for the primary objectives of the experiment:
(i) to assess the ability of p58C to participate in DNA charge transport and (ii) to examine
the differential effects of [4Fe4S] cluster oxidation state. The binding affinity of p58C for
the substrate should therefore not be particularly strong, so that any changes in affinity
between oxidized [4Fe4S]
3+
and reduced [4Fe4S]
2+
protein can be detected. A substrate
similar to the construct containing a 3
single-stranded DNA (ssDNA) overhang and 5
-
triphosphate, which Baranovskiy
et al.
(
9
) observe p58C binds tightly, would obscure the
observation of differences between the two redox states on the electrode. Moreover, as it
contains a 5
-ssDNA overhang, our electrochemistry substrate is similar to the primed ends
encountered by polymerase enzymes in cells.
Baranovskiy
et al.
additionally suggest that a mismatch in the nascent primer inhibits
initiation, but not truncation in the assay with a primed template substrate. On both well-
matched and mismatched substrates, we observe a mixture of initiation and elongation
products because primase binds to both ssDNA and primed DNA portions of the substrate
(
1
). Primase initiation products (7 to 29 nucleotides) are synthesized on the ssDNA template
used to generate a mismatched primer.
Baranovskiy
et al.
also criticize our assignment of the oxidized [4Fe4S]
3+
species generated
electrochemically. However, we show directly using cyclic voltammetry, as seen in figures 1
and 2 of (
1
), that we generate the oxidized [4Fe4S]
3+
product. The cathodic peaks in figure 1
and figure 2 (
1
) furthermore show the reduction of an oxidized species on the DNA-
modified electrode. Additionally, chemical oxidants with potentials similar to the potential
applied to p58C can oxidize human p58C to the [4Fe4S]
3+
state, as demonstrated by
electron paramagnetic resonance spectroscopy (
10
), supporting our assignment of the
[4Fe4S]
3+
species. We were not able to perform binding affinity measurements with the
electrochemically oxidized p58C because the oxidized protein is unstable over the long
periods of time required for the measurement.
Finally, Baranovskiy
et al.
criticize our performing in vitro priming assays under anaerobic
conditions that they argue do not correspond to the cellular environment. Anaerobic
conditions were used to ensure that we had full control over the redox state of the [4Fe4S]
cluster, which can be oxidized in the presence of air (
11
). In fact, controls performed under
aerobic conditions gave similar results overall, although with greater scatter and lower
precision.
Overall, the positioning of tyrosines 309, 345, and 347 in both structures of human p58C,
irrespective of local conformation, suggests a feasible pathway for electron transfer through
the [4Fe4S] protein. The electrochemical experiments with p58C variants, and the
biochemical activity assays with corresponding full primase variants, illuminate the electron
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transfer chemistry performed by the primase [4Fe4S] cluster and the effect of this chemistry
on DNA binding. Primer elongation assays with well-matched and mismatched template
strands, moreover, demonstrate the regulatory role of DNA charge transport in termination.
Thus, the concerns raised do not negate either our experimental observations of redox
chemistry performed by the [4Fe4S] cluster or the proposal of a role for the redox chemistry,
coupled to DNA charge transport, in regulating the activity of human DNA primase.
References
1. O’Brien E, et al. Science. 2017; 355:eaag1789. [PubMed: 28232525]
2. Baranovskiy AG, et al. Science. 2017; 357:eaan2396. [PubMed: 28729484]
3. Pellegrini L. Science. 2017; 357:eaan2954. [PubMed: 28729486]
4. Vaithiyalingam S, Warren EM, Eichman BF, Chazin WJ. Proc Natl Acad Sci USA. 2010;
107:13684–13689. [PubMed: 20643958]
5. Agarkar VB, Babayeva ND, Pavlov YI, Tahirov TH. Cell Cycle. 2011; 10:926–931. [PubMed:
21346410]
6. Sauguet L, Klinge S, Perera RL, Maman JD, Pellegrini L. PLOS ONE. 2010; 5:e10083. [PubMed:
20404922]
7. Winkler JR, Gray HB. J Am Chem Soc. 2014; 136:2930–2939. [PubMed: 24499470]
8. Baranovskiy AG, et al. J Biol Chem. 2016; 291:10006–10020. [PubMed: 26975377]
9. Baranovskiy AG, et al. J Biol Chem. 2016; 291:4793–4802. [PubMed: 26710848]
10. Weiner BE, et al. J Biol Chem. 2007; 282:33444–33451. [PubMed: 17893144]
11. Imlay JA. Mol Microbiol. 2006; 59:1073–1082. [PubMed: 16430685]
O’Brien et al.
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Fig. 1. Wild-type (WT) p48/p58 versus p48/p58Y309F activity on ssDNA
(A)
Gel separation of products for primase initiation assay comparing WT and charge
transfer–deficient primase.
(B)
Product quantifications for p48/p58Y309F, with WT primase
(p48/p58) products normalized to one. Values shown are the mean of
n
= 3 trials; error bars
represent standard deviation. *, 0.001 ≤
P
< 0.005; **,
P
< 0.001; Student’s
t
test. All
activity assays were performed under anaerobic conditions. Reactions contained 400 nM
primase variant, 250 nM ssDNA, 188 μM uridine triphosphate (UTP), 112 μM cytidine
triphosphate (CTP), 1 μM
α
−32
P adenosine triphosphate (ATP) in 50 mM Tris, pH 8.0, 3
mM MgCl
2
.
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