Yeast require redox switching in DNA primase
Elizabeth O
’
Brien
a,1
, Lauren E. Salay
b,c,d,1
, Esther A. Epum
e
, Katherine L. Friedman
e
, Walter J. Chazin
b,c,d,2
,
and Jacqueline K. Barton
a,2
a
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125;
b
Department of Biochemistry, Vanderbilt
University, Nashville, TN 37235;
c
Department of Chemistry, Vanderbilt University, Nashville, TN 37235;
d
Center for Structural Biology, Vanderbilt University,
Nashville, TN 37235; and
e
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235
Edited by JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA, and approved November 5, 2018 (received for review June 22, 2018)
Eukaryotic DNA primases contain a [4
Fe4S] cluster in the C-terminal
domain of the p58 subunit (p58C) that affects substrate affinity but is
not required for catalysis. We show that, in yeast primase, the cluster
serves as a DNA-mediated redox switch governing DNA binding, just
as in human primase. Despite a diffe
rent structural arrangement of
tyrosines to facilitate electron transfer between the DNA substrate
and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397
alters the same electron transfer chemistry and redox switch.
Mutation of conserved tyrosine 395 diminishes the extent of p58C
participation in normal redox-swi
tching reactions, whereas mutation
of conserved tyrosine 397 causes oxidative cluster degradation to the
[3Fe4S]
+
species during p58C redox signaling. Switching between
oxidized and reduced states in the presence of the Y397 mutations
thus puts primase [4Fe4S] cluster integrity and function at risk. Con-
sistent with these observations, we
find that yeast tolerate mutations
to Y395 in p58C, but the single-residue mutation Y397L in p58C is
lethal. Our data thus show that a constellation of tyrosines for
protein-DNA electron transfer m
ediates the redox switch in eukary-
otic primases and is required for primase function in vivo.
DNA replication
|
DNA charge transport
|
iron
–
sulfur proteins
D
NA replication requires the coordinated activity of several
polymerase enzymes (1). DNA primase begins replication on
the ssDNA template, synthesizing a short (8
–
12 nt) RNA primer
before handing off the primed template to DNA polymerase
α
.
Primase contains an RNA polymerase subunit (p48) and a regu-
latory subunit (p58) (2). Primase (3, 4) and many other, if not all,
replicative polymerases contain at least one [4Fe4S] cluster (5, 6),
a metal cofactor associated with biological redox chemistry (7).
Given the high metabolic expense associated with incorporation of
a cluster into one of these proteins (8), this cofactor is presumed
to play a functional role. Interestingly, the clusters in human DNA
primase (9) and yeast DNA polymerase
δ
(10) have been dem-
onstrated to perform DNA-mediated redox chemistry.
The [4Fe4S] cluster in eukaryotic primases is essential for
primer synthesis on the ssDNA template in vitro (3, 4) and in
cells (11). The cluster is located in the C-terminal domain of the
regulatory subunit, p58C, which is flexibly tethered to the N-
terminal domain (12, 13) and can bind DNA independently of
the rest of the primase enzyme (14, 15). In human DNA primase,
the cluster acts as a redox switch, modulating the interaction of
p58C with its substrate (9). Binding of the DNA polyanion shifts
the [4Fe4S] cluster redox potential, so that the [4Fe4S]
3
+
protein
is bound tightly, while the [4Fe4S]
2
+
protein is bound more
loosely. Endonuclease III in
Escherichia coli
similarly undergoes
an
∼
500-fold increase in DNA binding affinity upon oxidation to
the [4Fe4S]
3
+
state from the resting [4Fe4S]
2
+
state (16, 17).
We have proposed that the redox switch in human primase may
facilitate binding and substrate handoff, through DNA-mediated
charge transport (9). In our model, active primase is in the
[4Fe4S]
3
+
state, coupled into the
π
-stacked bases for redox sig-
naling. The p48 RNA polymerase domain and the p58C [4Fe4S]
cluster domain are in contact with the nascent RNA/DNA. When
the RNA primer reaches the appropriate length (8
–
14 nt), we
propose that polymerase
α
, in the [4Fe4S]
2
+
state, contacts the
RNA/DNA, becoming activated toward oxidation. Polymerase
α
sends an electron through the RNA/DNA duplex to reduce pri-
mase to the [4Fe4S]
2
+
state, and in so doing is converted to the
[4Fe4S]
3
+
state. Primase dissociates, and polymerase
α
is now
tightly bound, effectively executing a primer handoff.
During handoff in the redox-switching model, charge must travel
through duplex RNA/DNA and protein matrix (9, 16, 17). Bio-
chemical and structural evide
nce shows that p58C contacts DNA
through basic arginine and lysine residues (13, 18, 19), positioning
the cluster 25
–
30 Å from the substrate. Charge must travel from
the DNA to the cluster, through a
pathway within p58C. Charge-
transfer pathways in proteins often comprise aromatic residues,
which have comparatively low ionization energies and can transfer
charge (20, 21) through single-st
ep tunneling or electron hopping.
In human primase, for example, the redox switch is mediated by
conserved tyrosine residues, Y309, Y345, and Y347 in the p58C
domain. Substitution of these residues with phenylalanine produces
redox-deficient mutants, defective f
or initiation of primer synthesis.
Redox pathways through several proteins (22, 23) have been
characterized using Tyr
→
Phe mutations to perturb sites along the
electron-hopping pathway.
In yeast (
Saccharomyces cerevisiae
) primase, residues Y395
and Y397 are conserved and orthologous to Y345 and Y347 in
human primase (9). Although positioned differently than their
human orthologs within the respective p58C crystal structures,
they are spaced
∼
10
–
15 Å apart, and could feasibly comprise
a redox pathway (20, 21). We hypothesized that yeast and hu-
man p58C have similar redox-switch mechanisms, despite their
different tyrosine constellations. Establishing that a yeast pri-
mase redox pathway exists also provides an opportunity to assay
Significance
Redox switching driven by [4Fe4S] cluster cofactors modulates
DNA binding affinity in proteins, providing a rapid, efficient
method of substrate binding and dissociation. Our study es-
tablishes an essential redox switch with an aromatic pathway
through the yeast DNA primase; a single-residue mutation at
position 397 along this redox pathway causes [4Fe4S] cluster
degradation and is lethal in yeast.
Author contributions: E.O., L.E.S., K.L.F., W.J.C., and J.K.B. designed research; E.O., L.E.S.,
and E.A.E. performed research; E.O., L.E.S., E.A.E., K.L.F., W.J.C., and J.K.B. analyzed data;
and E.O. and J.K.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This open access article is distributed under
Creative Commons Attribution-NonCommercial-
NoDerivatives License 4.0 (CC BY-NC-ND)
.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank,
www.rcsb.org
[PDB ID codes: WT p58C (
Saccharomyces cerevisiae
),
6DI6
; Y395F (
S. cerevisiae
),
6DTV
; Y395L (
S. cerevisiae
),
6DU0
; Y397F (
S. cerevisiae
),
6DTZ
; and Y397L (
S. cerevisiae
),
6DI2
].
1
E.O. and L.E.S. contributed equally to this work.
2
To whom correspondence may be addressed. Email: walter.j.chazin@vanderbilt.edu or
jkbarton@caltech.edu.
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1810715115/-/DCSupplemental
.
Published online December 12, 2018.
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the viability of yeast cells expressing redox-deficient primase
variants.
Results
Redox Switch Through a Tyrosine Pathway in Yeast p58C.
Human and
yeast p58C have similar overall structures (Fig. 1) with a backbone
root mean squared dev
iation (rmsd) of only 0.82 Å. Both proteins
bind DNA with micromolar affinity (14, 15). Yeast and human
p58C each contain a [4Fe4S] cluster buried within the protein,
∼
25
Å from the DNA binding interface (14, 15) (Fig. 1). Despite having
only 40% identical sequences, yeast and human p58C both have
multiple conserved tyrosines between the cluster and the DNA
binding interface. In human p58C, these tyrosines shuttle charge
through the protein to mediate the redox switch (9) (Fig. 1).
We sought to determine whether the yeast p58C [4Fe4S]
cluster has a redox-switching capability similar to that of human
p58C. We identified several tyrosine residues conserved in yeast
p58C, which are within feasible charge-transport distance of the
yeast p58C cluster or DNA-binding interface, as possible medi-
ators of a redox switch. Residues Y395 and Y397 in yeast p58C
(Fig. 1), orthologous to Y345 and Y347, respectively, in the
human p58C redox pathway, are prime candidates for a redox
role (9). Distances between tyrosine centroids are larger on av-
erage in the yeast protein crystal structure (12.9
–
15.3 Å) (14)
than in the human protein crystal structure (5.1
–
10.4 Å) (15), but
fall securely within the distance for feasible microsecond elec-
tron transfer. As the distance between centroids, rather than the
relative positions of tyrosines, is the primary factor determining
the feasibility of charge transfer through protein (21), we pre-
dicted that both pathways could mediate redox switching.
To test whether a change in the oxidation state of the [4Fe4S]
cluster in yeast p58C affects DNA binding, we compared anaer-
obically the electrochemical behavior of oxidized [4Fe4S]
3
+
p58C
and reduced [4Fe4S]
2
+
p58C on multiplexed DNA electrodes
(Fig. 1), modified with a 20-nt DNA duplex substrate containing a
3-nt, 5
′
- ssDNA overhang (
SI Appendix
, Table S1
) identical to the
substrate used to study human p58C (9). Purified WT yeast p58C,
in the absence of an applied potential, displays no redox signal in
cyclic voltammetry (CV) (
SI Appendix
,Fig.S1
). This form of the
protein is thus not coupled to the DNA base pairs for redox sig-
naling. WT p58C was then electrochemically oxidized by applying
a positive potential (412 or 512 mV vs. normal hydrogen electrode
(NHE); see
SI Appendix
, Table S2
) or electrochemically reduced
by applying a negative potential (
−
188 mV vs. NHE) to the electrode
surface. Bulk electrolysis was performed on individual electrodes
for a total of 8.3 min, to optimize the yield of electrochemically
converted protein. Bulk oxidation of a p58C sample, compared
with a buffer control, is shown in
SI Appendix
,Fig.S2
.
Oxidized or reduced p58C samples were scanned by CV im-
mediately after bulk electrolysis. The electrochemically oxidized
sample displays a large cathodic peak near
−
130 to
−
150 mV vs.
NHE in Tris storage buffer (20 mM Tris, pH 7.2, 75 mM NaCl).
After a single scan to negative, reducing potentials, the signal
disappears, indicating a loss of coupling between the [4Fe4S]
cluster and the DNA bases. (Fig. 2). Electrochemically reduced
p58C, conversely, does not display any signal after bulk elec-
trolysis. This result suggests that oxidized [4Fe4S]
3
+
yeast p58C is
tightly bound to DNA and electronically coupled into the bases
for redox signaling, but reduced [4Fe4S]
2
+
yeast p58C is loosely
associated and not coupled to the DNA for signaling.
Human p58C can be electrochemically converted from the oxi-
dized, tightly bound state to the reduced, loosely associated state
(9). Iterative oxidations on a DNA electrode indicate a similar
behavior in yeast p58C (Fig. 2). Oxidation of yeast p58C to the
[4Fe4S]
3
+
state produces a large, reduc
tive CV signal, indicating
conversion to the resting (3, 4) [4Fe4S]
2
+
state. The signal disap-
pears in the second CV scan, but a second oxidation regenerates the
reductive peak. A single electron t
ransfer reaction thus facilitates
conversion between two forms of p58C with dramatically different
DNA-binding and redox-signaling properties.
We next investigated whether the observed yeast p58C redox
switch is dependent on a pathway of
conserved tyrosine residues, as
in the human protein. We constructed yeast p58C variants with
mutations at Y395 or Y397. As these residues are conserved
orthologs to components of the human primase redox pathway,
they were prime candidates for the yeast primase redox pathway.
All generated p58C variants (Y395F, Y397F, Y395L, and Y397L)
load the [4Fe4S] cluster comparably to WT, as assessed by the ratio
of absorption at 410 nm/280 nm in UV-visible spectroscopy (
SI
Appendix
,Fig.S3
). Fluorescence anisotropy assays on WT and
mutant p58C (
SI Appendix
,Fig.S4
) show that all variants bind the
substrate with virtually the same low micromolar affinity. This result
suggests that the same number of p58C molecules are bound to the
DNA on the electrode surface before electrochemical oxidation,
when the sample is present largely in the [4Fe4S]
2
+
state. Since the
redox pathway through p58C is the conduit through which cluster
oxidation occurs, we expect that fewer mutants versus WT are
bound tightly to DNA in the [4Fe4S]
3
+
state after bulk oxidation, if
these tyrosines mediate the redox switch in yeast primase.
Analysis of charge transfer in the reductive peak (Q
CV
,
SI Ap-
pendix
, Table S2 and Figure S5
) for WT yeast p58C, p58C Y395F
(
SI Appendix
,TableS2
), Y397F (Fig. 2), Y397L (Fig. 3), and Y395L
(
SI Appendix
,Fig.S7
) demonstrates that the mutants are consis-
tently redox-deficient relative to WT p58C. It is interesting to ob-
serve however that the p58C variants, particularly the Tyr
→
Phe
mutants, retain partial redox-switc
hing ability. Phenylalanine resi-
dues are generally less capable of mediating electron transfer
through protein than tyrosines, due to their higher ionization energy
(20) and inability to form discrete cation radicals; phenylalanines,
however, can mediate electron transfer, although less efficiently
through tunneling (22), partially inhibiting charge transfer in some
protein systems (23, 24).
Tyrosine Mutations Do Not Change Yeast p58C Structure.
X-ray
crystal structures of the WT an
d mutant yeast p58C demonstrate
that no changes in the overall structure of the protein are caused by
the tyrosine mutations (Fig. 4 and
SI Appendix
,TableS3
). Overlays
of WT yeast p58C and p58C Y395F, Y397F, Y395L, and Y397L, all
of which have an rmsd less than 0.5 Å from the WT, underscore the
structural similarity. The F395 and F397 aromatic rings in Y395F
and Y397F, respectively, are oriented in almost exactly the same
position as the aromatic rings in th
e tyrosines of the WT protein, as
Fig. 1.
Yeast and human p58C structures can both support a redox switch.
(
Left
) Comparison of p58C structures (
Upper
) and conserved tyrosines
(
Lower
) near the [4Fe4S] cluster in yeast p58C (light brown) and human p58C
(dark brown). Structures rendered using human p58C [Protein Data Bank
(PDB) ID code 3L9Q] (15) and yeast p58C (PDB ID code 6DI6,
SI Appendix
,
Table S3
) structures. (
Right
) Diagram of the multiplexed DNA electrochem-
istry platform (
Upper
) and a cartoon depicting the change in DNA binding
associated with redox switching (
Lower
). Human p58C image adapted from
ref. 15. Multiplex chip and yeast p58C images from ref. 9. Reprinted with
permission from
AAAS
.
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CHEMISTRY
BIOCHEMISTRY
had been seen previously wi
th human p58C Y345F and Y347F
mutants (9). Notably, the more drastic substitution of the tyrosine
aromatic ring with the leucine aliphatic chain also has very little
effect on the structure, including the orientation of side chains.
The similar WT and mutant yeast p58C structures and substrate
affinities corroborate that the dif
ference in redox signal between WT
and mutant p58C is a result of diff
erences in the redox-switching
ability and not the ability to interact with a DNA substrate. All p58C
variants were crystallized, moreo
ver, with high [4Fe4S] cluster
loading in the protein sample, supported by the UV-visible spectra
(
SI Appendix
,Fig.S3
). Overall, we observe that the p58C mutant
proteins are less effective in part
icipating in redox signaling on DNA
relative to WT, when equal amounts of [4Fe4S] protein (determined
spectroscopically) are initially deposited on the Au electrode. Dif-
ferent electrochemical behavior o
f these variants is thus a conse-
quence of different chemica
l/electronic properties.
Reversible, Nucleotide Triphosphate-Dependent Redox Activity in
Yeast p58C.
Yeast p58C displays a semireversible, nucleotide
triphosphate (NTP)-dependent redox signal on DNA under an-
aerobic conditions, centered at 149
±
14 mV vs. NHE, in the
presence of 1.25 mM ATP. This potential is within the biological
redox potential range (25) and comparable to the potential values
observed for other DNA-processing [4Fe4S] proteins, suggesting
that active primase may signal other DNA-bound [4Fe4S] proteins
during replication (10, 26
–
28).ThesignalobservedinCVis71
±
10% reversible, displaying an average charge-transfer value of
8.7
±
4 nC in the reductive peak and 5.8
±
2 nC in the oxidative
peak. The larger cathodic wave in CV is consistent with the oxi-
dized [4Fe4S]
3
+
protein having a higher binding affinity and being
more strongly coupled to the DNA bases for redox activity than the
reduced [4Fe4S]
2
+
protein. The reductive peak corresponds to the
electrochemical conversion of [4Fe4S]
3
+
p58C to [4Fe4S]
2
+
p58C,
and the oxidative peak is a measurement of the reverse process.
The primase redox-switch model predicts that more oxidized
[4Fe4S]
3
+
protein is bound in the p58C:DNA:NTP complex than
reduced [4Fe4S]
2
+
protein. The data corroborate this model and
suggest that NTP binding induces redox switching in yeast p58C.
To assess whether this reversible signal depends on the p58C
charge-transfer pathway, we measured the NTP-dependent signals
of yeast p58C Y395 and Y397 mutants. Unlike WT yeast p58C, no
mutants generated a signal detectable by CV in the presence of
1.25 mM ATP. We therefore used square-wave voltammetry
(SWV), an electrochemical technique which can reliably detect
smaller signals (29) to aid in chara
cterizing mutant NTP-dependent
redox activity. We observe a single reductive peak in SWV at
−
86
±
13 mV vs. NHE and
−
90
±
4 mV vs. NHE, for Y397F and Y397L,
respectively, on a DNA electrode in the presence of 1.25 mM ATP
(Fig. 5). This signal appears at a potential distinct in SWV from the
semireversible signal for the WT p58C (
+
85
±
9mVvs.NHE),
reflecting the presence of a different species (Fig. 5).
We next tested whether the p58C mutants displayed any redox
activity in the presence of 2.5 mM ATP. The Y395L mutant
displayed no measurable redox signal in the presence of 1.25 or
2.5 mM ATP (
SI Appendix
, Fig. S8
). The Y395F variant displays
some reversible charge transport centered at 162
±
6 mV vs.
NHE in the presence of 2.5 mM ATP, although the charge
transport (3
±
1 nC in the cathodic peak, 2
±
0.6 nC in the anodic
peak) is diminished relative to WT with 1.25 mM ATP (
SI Ap-
pendix
, Fig. S9
). In the presence of 2.5 mM ATP, Y397F displays
an irreversible peak at a similar potential to the peak observed
with 1.25 mM ATP (
SI Appendix
, Fig. S10
) in some but not all
Fig. 2.
The yeast p58C redox switch is mediated by conserved tyrosines. (
Left
) Bulk electrolysis and CV of WT yeast p58C. (
Center
) Bulk electrolysis and CV of
p58C Y397F. (
Right
) Iterative electrochemical oxidation regenerates a CV signal on DNA. Cartoon (
Bottom Right
) Illustration of the electrochemically re-
versible redox switch in yeast p58C. All scans were performed anaerobically, on 30
μ
M [4Fe4S] p58C (WT, Y395F, or Y397F), 20 mM Tris, pH 7.2, 75 mM NaCl,
100-mV/s scan rate for CV. Illustration from ref. 9. Reprinted with permission from
AAAS
.
Fig. 3.
Tyrosine to leucine p58C mutants are redox-deficient. (
Left
) Bulk
electrolysis and CV of WT yeast p58C. (
Right
) Bulk electrolysis and CV of yeast
p58C Y397L. The mutant displays decreased redox activity in CV. All scans
were performed anaerobically, on 57
μ
M [4Fe4S] p58C (WT or Y397L), 20 mM
Hepes, pH 7.2, 75 mM NaCl, 100-mV/s scan rate for CV.
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scans. The Y397L variant displays minimal reversible redox ac-
tivity in CV (
SI Appendix
, Fig. S11
) centered at 140
±
17 mV vs.
NHE, with charge-transport values of 1.7
±
1 nC in the cathodic
peak and 1.5
±
1 nC in the anodic peak. The NTP-dependent
redox activity of the tyrosine mutants is thus consistently di-
minished relative to WT p58C, suggesting the primary redox
pathway through Y395 and Y397 is important for redox signaling
when bound to all necessary substrates for primer synthesis.
This irreversible reductive peak between
−
80 and
−
90 mV vs.
NHE likely corresponds to the [3Fe4S]
+
degradation product of
the [4Fe4S] cofactor (30). This irreversible secondary reductive
peak is actually observed in WT yeast p58C, alongside the pri-
mary wave, in the presence of a large excess of ATP (
SI Ap-
pendix
, Fig. S12
). A signal in the reductive wave at comparable
potentials was recently observed electrochemically and spectro-
scopically in a cancer-causing variant of human base excision
repair protein MUTYH, when exposed to atmospheric oxygen
(31). The [3Fe4S]
+
degradation product is a consequence of
oxidation from the resting [4Fe4S]
2
+
state to the [4Fe4S]
3
+
state
(26, 31) in a mutant with a destabilized metal cofactor. The
[3Fe4S]
+
product can also occur as a result of oxidation in an
aerobic atmosphere during purification or sample preparation
(6, 26). Since the [3Fe4S]
+
/0
reduction peak occurs in p58C
Y397F/Y397L in the presence of NTPs and in the absence of
oxygen, however, these mutants likely undergo some electron
transfer when bound to DNA and NTPs, anionic substrates
expected to shift the potential of the [4Fe4S] cluster (25, 32).
Since the compromised redox pathway inhibits reversible cycling
between the [4Fe4S]
2
+
and [4Fe4S]
3
+
states, redox signaling in
these p58C variants can lead to a
“
trapped
”
high-energy
[4Fe4S]
3
+
species which cannot easily be reduced back to the
[4Fe4S]
2
+
state; the cluster then becomes unstable and degrades.
Efficient redox switching is essential for regulation of activity, as
well as [4Fe4S] cluster stability.
The Yeast p58C Redox Switch Is Necessary for Viability.
After char-
acterizing the redox switch in yeast DNA primase, we sought to
investigate the consequences of this chemistry in yeast cells. Both
subunits of yeast primase are essential; partially defective alleles
compromise DNA synthesis and cell growth (33, 34). Liu and
Huang (11) additionally have shown that mutations of the cys-
teine residues ligating the p58C [4Fe4S] cluster cause growth
defects in cells, suggesting the importance of the primase cluster
for viability. We have shown that mutations at p58 residues Y395
or Y397 along the redox pathway impair the Fe
–
S redox switch
in vitro and hypothesized that these mutations would compromise
cell growth. Since we observe the formation of a putative [3Fe4S]
+
species in the presence of DNA and NTPs electrochemically for
the Y397 mutants, we were interested in potential differences
between Y395 and Y397 tyrosine pathway mutants. Single-site
mutations were introduced into the chromosomal gene encoding
the p58 subunit of
S
.
cerevisiae
DNA primase (
PRI2
, ref. 34) at
positions Y395 and Y397 under control of the endogenous pro-
moter. We incorporated Y395F, Y395L, Y397F, or Y397L muta-
tions into the yeast genome to investigate the biological effects of
both mutations at different loci along the charge-transfer pathway
and mutations to aromatic phenylal
anine versus aliphatic leucine. As
these redox-pathway mutations do not affect [4Fe4S] cluster loading
or DNA binding of p58C in vitro, we could specifically assess the
effects of the cluster redox switch on cellular fitness.
We investigated whether the Y395F and Y397F mutations in
p58C, which retain some redox switching on DNA electro-
chemically (Fig. 2 and
SI Appendix
, Fig. S5
), affect cellular fit-
ness. Haploid strains expressing the
pri2Y395F
and
pri2Y397F
alleles were viable at 30 °C and grew comparably to the parental
PRI2
strain (
SI Appendix
, Fig. S13
). The Tyr
→
Phe mutants re-
tain some redox-switching activity in the electrochemical scans, and
the viability of cells is consistent with a partially but not completely
inhibited redox pathway in these variants. Phenylalanine has an
aromatic side chain, and although the ionization energy of this
residue is higher than that of tyrosine (20), phenylalanine is still
found disproportionately in oxidoreductase enzymes, suggesting
that it can aid in redox pathways through protein (21).
We next investigated the effects of the
pri2Y395L
and
pri2Y397L
mutations on yeast viability. As aliphatic leucine more
strongly abrogates the redox pathway through p58C (Fig. 3 and
SI Appendix
, Fig. S7
), we expected to observe more severe
phenotypes with Tyr
→
Leu mutations in
PRI2
than with Tyr
→
Phe mutations. We constructed a haploid
pri2Y395L
strain and
observed, as with the Tyr
→
Phe variants, no growth defect rel-
ative to WT
PRI2
strains. In contrast, we were unable to con-
struct a haploid strain containing the
pri2Y397L
allele. To confirm
Fig. 5.
Reversible, NTP-dependent redox switching in yeast p58C. (
Left
)Car-
toon depicts DNA- and NTP-bound yeast p58C cycling between [4Fe4S]
3
+
and
[4Fe4S]
2
+
states. (
Right
) CV scans of WT yeast p58C (blue), p58C Y397F (green),
and p58C Y397L (red) in the presence of 1.25 mM ATP. WT p58C displays a
signal at 149
±
14 mV vs. NHE. Reductive SWV of WT and mutant p58C. A small
signal near
−
80 to
−
90 mV vs. NHE appears for Y397F and Y397L. All scans
were performed anaerobically, on 57
μ
M [4Fe4S] p58C variant and 1.25 mM
ATP in 20 mM Hepes, pH 7.2, 75 mM NaCl, 100-mV/s scan rate for CV, 15-Hz
frequency, 25-mV amplitude, 60-mV/s scan rate for SWV. Potentials reported
are mean
±
SD of at least three trials. The increase in current at
−
0.3 V vs. NHE
appears in the buffer scan and is p58C-independent.
Fig. 4.
Protein structure is preserved upon mutation of Y395 and Y397 in
yeast p58C. The WT yeast p58C (brown) crystal structure is overlaid with each
redox-deficient mutant. Mutation of tyrosines to aromatic phenylalanine or
aliphatic leucine minimally perturbs protein structure.
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lethality of
pri2Y397L
, we transformed the WT haploid strain with
a
URA3
-marked
PRI2
complementing plasmid and subsequently
integrated either WT or mutant
PRI2
alleles at the endogenous
locus. Complemented strains expressing WT
PRI2
,
pri2Y397F
,or
pri2Y397L
grew well on media lacking uracil, as expected. How-
ever, when plated on media containing 5-fluoroorotic acid
(5-FOA), only the
pri2Y397L
strain failed to generate colonies
(Fig. 6). Since the protein product of the
URA3
gene converts
5-FOA to toxic 5-fluorouracil, this result confirms that cells
expressing the lethal
pri2Y397L
allele cannot tolerate loss of the
URA3
-marked
PRI2
complementing plasmid. Phenotypes of the
pri2Y395L
and
pri2Y397L
mutants were confirmed by tetrad
analysis (
SI Appendix
,Fig.S14
A
).
While the
pri2Y397L
mutation has no discernible effect on the
protein structure in vitro (Fig.
4), we wanted to confirm that the
mutant is expressed and associates with the primase complex in
yeast. If so, we would predict that the Y397L mutation in
PRI2
impairsyeastgrowthwhenoverexpressedinanotherwiseWT
background, competing with the normal protein for incorporation
into the primase complex. Conditional overexpression of the Y397L
variant, but not of
WT PRI2
, causes severe growth defects (
SI Ap-
pendix
,Fig.S14
B
). The Y397L mutation thus generates a p58 protein
that can associate with primase, but is severely impaired in vivo.
The severe phenotype in the
pri2Y397L
strain is consistent with
the effect of the Y397L mutation on p58C redox signaling activity
(Figs. 3 and 5). This mutant displays minimal redox switching on
DNA, and the signal in the presence of DNA and NTPs suggests
that redox signaling leads to oxidative degradation of the cluster in
p58C Y397L. Small-molecule reactive oxygen species such as hy-
drogen peroxide can damage the [4Fe4S] cluster directly, causing
degradation to the [3Fe4S]
+
form (30), or indirectly through DNA
charge transport. We have observed, for example, that guanine
radicals in oxidized duplex DNA induce redox switching in a
bound [4Fe4S] Endonuclease III protein (35). WT p58C can more
easily cycle between the [4Fe4S]
2
+
and [4Fe4S]
3
+
states than the
tyrosine mutants when bound to both DNA and NTPs, mimick-
ing the active primase. Y395F and Y395L variants appear to
lose redox-switching activity, but are not oxidatively degrading
on the DNA electrode. The Y397F and Y397L mutants, con-
versely, appear to be oxidized from the [4Fe4S]
2
+
state to the
[4Fe4S]
3
+
state, but they are not easily reduced back to the
[4Fe4S]
2
+
state, causing the reductive peak likely associated with
the irreversible [3Fe4S]
+
/0
couple on DNA (31) to appear. We
observe therefore that the combination of greater redox attenua-
tion overall in Tyr
→
Leu variants (Fig. 3 and
SI Appendix
, Fig.
S7
), and the oxidative degradation observed in Y397F/L variants
(Fig. 5), dysregulate primase activity to a point for which cellular
machinery cannot compensate.
Discussion
Reversible, redox-driven switches control DNA binding affinity in
[4Fe4S] repair and replication enzymes, facilitating rapid binding
and dissociation (9, 25, 32). The oxidized [4Fe4S]
3
+
bacterial
glycosylase Endonuclease III, for example, binds the DNA poly-
anion 550-fold more tightly than the reduced [4Fe4S]
2
+
Endo-
nuclease III. Reversible cluster oxidation and reduction facilitates
DNA-mediated redox signaling between Endonuclease III and
other [4Fe4S] repair proteins in the first steps of locating oxidative
DNA damage (32). The [4Fe4S] enzyme yeast DNA polymerase
δ
is DNA-bound and active in the [4Fe4S]
2
+
state, when associated
with proliferating cell nuclear antigen (PCNA) (36). When oxi-
dized to the [4Fe4S]
3
+
state, however, PCNA-associated poly-
merase
δ
binds DNA even more tightly, stalling replication (10).
This change in binding may allow polymerase
δ
to sense and re-
spond to oxidative stress. Similar redox switching chemistry thus
regulates diverse, specialized DNA-processing [4Fe4S] enzymes.
Here we establish a redox switch, driven by a change in [4Fe4S]
cluster oxidation state, that regulates DNA binding and redox
signaling in eukaryotic DNA primase. This switch regulates primer
synthesis, but not catalytic activity, in human primase (9). Struc-
tural and biochemical evidence suggests that primase adopts a
compact configuration during activity, with both the p48 (RNA
polymerase) subunit and the p58C [4Fe4S] domain contacting the
∼
8
–
14-nt RNA/DNA duplex substrate (13, 18, 19). Redox path-
ways comprising conserved tyrosines spaced 10
–
15 Å apart, shuttle
charge approximately
∼
25
–
30 Å from the DNA binding interface
to the [4Fe4S] cluster. Aromatic tyrosines spaced
≤
15 Å apart can
facilitate microsecond electron transfer (20, 21) in protein, pos-
sibly through formation of hopping intermediates. Despite pre-
vious arguments that structural differences in yeast and human
primase preclude a general redox role for these residues (37), the
electrochemical and biological data unequivocally demonstrate
that the electron-transfer pathway is conserved. Electrochemical
attenuation of the p58C redox signal on DNA and lethality in
yeast due to a single-residue redox-pathway mutation suggest a
more significant role than the contributions of these tyrosines to
any network of p58C/substrate hydrogen bonds (13).
The primary redox pathway affecting the yeast p58C [4Fe4S]
cluster redox switch likely involves Y395 and Y397. When these
conserved residues are mutated, two possible events may occur.
First, the mutant residue at posit
ion 395 or 397 may transfer charge
through single-step tunneling, as opposed to electron hopping onto
the tyrosine. Additionally, the mutation may route the electron
through another primary hopping pathway, less efficient than the
WT, within p58C. Multiple potential pathways through p58C, in-
volving conserved residues Y431 and Y352 for example, are
available to transfer charge between bound DNA and the cluster.
Our model for redox-driven pri
mer handoff is compatible with
the time scale of primer synthesis. Studies of calf thymus primase
(38) suggest that primer synthesis
is quite slow, with a first-order
rate constant of 0.0027 s
−
1
. Picosecond DNA-mediated electron
transfer (39), microsecond electr
on transfer through primase/poly-
merase-
α
protein matrix (21), and microsecond/millisecond con-
formational changes in the polymerase-
α
active site (40) would all
occur securely within this time. Th
e aerobically measured binding
constants of eukaryotic DNA primase and polymerase
α
(
∼
100
nM
–
1
μ
M) (9, 14, 41) interestingly correspond to the upper limit of
the estimated primase/polymerase-
α
concentration in the yeast
nucleus, based on protein copy number (42) and yeast cell volume
(43) estimates. Tighter protein-DNA binding than these
Fig. 6.
Yeast p58 (Pri2) Y397L mutation is lethal. Haploid yeast were
transformed with a
URA3
-marked complementing plasmid expressing
PRI2
.
The indicated
PRI2
alleles (
PRI2
WT,
Y397F
,or
Y397L
) were introduced by
integration at the chromosomal locus (
Materials and Methods
). After veri-
fication of the genotype, individual transformants were streaked on media
lacking (
Upper
) or containing (
Lower
) 5-FOA to select for cells that have lost
the complementing plasmid.
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O
’
Brien et al.
numbers indicate, which redox switching provides a means to
access, may be necessary to efficiently execute replication.
Redox signaling in eukaryotic DNA primase is mediated by an
aromatic pathway in the primase [4Fe4S] domain. Mutation of a
conserved aromatic tyrosine at position 395 attenuates redox-
switching activity driven by the primase [4Fe4S] cluster. Muta-
tion of conserved tyrosine 397 attenuates redox switching and
leads to oxidative degradation during redox signaling on DNA.
The Y397L mutation abrogates the aromatic pathway and leads
to cluster degradation during the redox switch, conferring le-
thality in yeast. Tyrosines 395 and 397 are not located near the
primase catalytic site, yet a mutation at a position between the
[4Fe4S] cluster and DNA-binding domain affects the regulatory
redox switch and can prohibit cell growth. These observations
support our proposal that the conserved redox chemistry of the
[4Fe4S] cluster in DNA primase plays a central role in co-
ordinating the initial steps of eukaryotic DNA priming.
Materials and Methods
DNA Modified Electrode Assembly/Preparation.
DNA and multiplexed chips
were prepared and cleaned as described in
SI Appendix
. Assembled chips
were transported into an anaerobic glove bag (Coy Products) and washed
with deoxygenated protein storage buffer.
Sample Preparation for Electrochemistry.
Yeast p58C and tyrosine variants
were overexpressed and purified as described in
SI Appendix
. Tyrosine mu-
tagenesis and characterization of p58C variants is described in
SI Appendix
.
Samples were buffer exchanged if necessary through a procedure detailed in
SI Appendix
. Samples were deposited onto multiplex chip quadrants, then
bulk solution was added to a final volume of 200
–
300
μ
L.
WT/Mutant p58C Electrochemistry.
All electrochemistry was performed using a
CHI620D potentiostat and 16-channel multiplexer (CH Instruments), in an
anaerobic glove chamber. Details of measurements under specific conditions
can be found in
SI Appendix
.
X-Ray Crystallography.
The protein was dialyzed into 20 mM Hepes (pH 6.8),
2 mM DTT, and 75 mM NaCl and concentrated to
∼
5 mg/mL. Details of crystal
formation and data collection/analysis can be found in
SI Appendix
.
Yeast Strain Construction.
PRI2
mutations were introduced into haploid strain
YKF201 (
MAT
a
trp1 leu2 ura3 his7
) by two-step gene replacement (44).
Detail of this procedure and the procedure used to overexpress PRI2 alleles
can be found in
SI Appendix
.
ACKNOWLEDGMENTS.
We are grateful to the NIH for Operating Grants R01
GM126904 (to J.K.B.), R35 GM40120 (to W.J.C.), and R01 GM123292 (to K.L.F.), and
Training Grants T32 GM07616 (to E.O.) and T32 GM08230 (to L.E.S.). We received
additional support from the Moore Foundation (J.K.B.) and a Ralph M. Parsons
Fellowship (to E.O.). Diffraction data were collected at the Advanced Photon
Source, operated by the US Department of E
nergy (Contract DE-A
C02-06CH11357).
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O
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PNAS
|
December 26, 2018
|
vol. 115
|
no. 52
|
13191
CHEMISTRY
BIOCHEMISTRY