of 7
Supporting Information
:
Substrate Binding Regulates Redox Signaling in Human DNA
Primase
Elizabeth O’Brien,
1,2
Marilyn E. Holt,
3
Lauren E. Salay,
3
Walter J. Chazin,
3*
and
Jacqueline
K. Barton
1*
Contents:
Figure S1
. CV of Electrochemically unaltered human p48/p58.
Figure S2
. UV
-
Visible spectrum of p48/p58.
Figure S3.
Activity Gel showing Substrate Binding Order Does not Affect Initiation.
Figure S4
. Quantification of primase
initiation products in the pre
-
incubation assay.
Figure S5
. Electrostatic fields at the surface of p48 and p58C with a DNA substrate bound.
Table S1.
DNA substrates used for electrochemistry and DNA primase activity assays.
Figure S1
.
Electrochemically unaltered human p48/p58 displays no significant redox activity
in CV scans, suggesting that the resting state of the enzyme is either not bound to DNA or
bound in a manner that does not promote coupling of the [4Fe4S] cluster to the DNA b
ases for
redox signaling. A small peak associated putatively with the [3Fe4S]
+
degradation product is
observable using the more sensitive electrochemical technique, Square Wave Voltammetry
(SWV), but no significant redox activity from the [4Fe4S]
2+/3+
c
oup
le
is observed.
S1
S
2
4
3
2
1
0
200
300
400
500
600
700
800
Wavelength (nm)
Figure S2
. UV
-
Visible spectrum of p48/p58 after exchange into 20mM HEPES, pH 7.2,
150mM NaCl
, 5% glycerol from the original Tris storage buffer.
Absorbance (A.U.)
S
3
Figure S3.
Substrate Binding Order Does not Affect Primase Initiation
.
Gel separation of
products for three primase initiation reactions. Primase alone (p48/p58
only, grey)
was added
to DNA and NTPs
, primase pre
-
incubated with NTPs was added to DNA (p48/p58 + NTPs,
green), or primase pre
-
incubated with DNA was added to NTPs (p48/p58 + DNA, blue) to
start the reaction. Pre
-
incubation times were 30 minutes in ana
erobic conditions; all pre
-
incubation volumes were equal. All experiments were performed in anaerobic conditions, with
400 nM p48/p58, 250nM primed DNA, 188
μ
M [UTP], 112
μ
M [CTP], 1
μ
M
α
-
32
P ATP in
50mM Tris, pH 8.0, 3mM
MgCl
2,
37°C.
S
4
Incubation Time
(minutes,
37°C)
Incubation Time (minutes,
37°C)
Figure S4
. Quantification of primase initiation products in the pre
-
incubation assay. Total
products (left) and primer
-
length (7
-
10nt) products (right) do not differ within error for the
maj
ority of time points assayed in the pre
-
incubation reaction under anaerobic conditions.
This similarity suggests that substrate binding alone does not drive primase initiation, though
it appears to drive elongation activity. All measurements are mean
+
S.D
. for n
= 3 trials.
S
5
Figure S5
. Electrostatic fields at the surface of p48 and p58C with a DNA substrate
bound. The p48 catalytic site is indicated by a yellow asterisk. The black ring on p48
indicates where the DNA substrate and p58C need
to be positioned for the initiation of
priming. Coordinates used: p48 (4LIL); p58C
-
substate (5F0Q). Figure was generated
using the MSMS package in UCSF Chimera (https://doi.org/10.1002/jcc.20084)
S
6
Table S1.
DNA substrates used for electroc
hemistry and DNA primase activity assays.
Electrochemistry of p48/p58 and p48/p58 was performed on self
-
assembling monolayers of a
36
-
mer DNA duplex substrate with a 9
-
nt 5’
-
ssDNA overhang. A 50
-
nt ssDNA substrate with
a single thymine base complementary
to the
α
-
32
P radiolabeled ATP was used in the primase
initiation assay. 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.
U
=
2’
-
OMe rU, SH =
-
(CH
2
)
6
-
SH.