Articles
https://doi.org/10.1038/s41557-018-0068-x
© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
A human MUTYH variant linking colonic polyposis
to redox degradation of the [4Fe4S]
2
+
cluster
Kevin J. McDonnell
1,9
, Joseph A. Chemler
2,9
, Phillip L. Bartels
3,9
, Elizabeth O’Brien
3
,
Monica L. Marvin
4
, Janice Ortega
5
, Ralph H. Stern
6
, Leon Raskin
7
, Guo-Min Li
5
,
David H. Sherman
2,8
*, Jacqueline K. Barton
3
* and Stephen B. Gruber
1
*
1
University of Southern California Norris Comprehensive Cancer Center, Los Angeles, CA, USA.
2
Life Sciences Institute, University of Michigan, Ann Arbor,
MI, USA.
3
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
4
Department of Human Genetics,
University of Michigan, Ann Arbor, MI, USA.
5
Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
6
Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
7
Amgen Inc., Thousand
Oaks, CA, USA.
8
Departments of Medicinal Chemistry, Chemistry and Microbiology & Immunology, University of Michigan, Ann Arbor, MI, USA.
9
These authors contributed equally to this work: Kevin J. McDonnell, Joseph A. Chemler, Phillip L. Bartels. *e-mail: davidhs@umich.edu;
jkbarton@caltech.edu; sgruber@med.usc.edu
SUPPLEMENTARY INFORMATION
In the format provided by the authors and unedited.
NATURE CHEMiSTRY
|
www.nature.com/naturechemistry
Page |
1
Supplementary
Information
A Human MUTYH Variant Linking Colonic Polyposis to Redox
Degradation of the [4Fe4S]
2+
Cluster
Kevin J. McDonnell
1,†
, Joseph A. Chemler
2,†
, Phillip L. Bartels
3,†
, Elizabeth O’Brien
3
, Monica L.
Marvin
4,5
, Janice Ortega
1
, Ralph
H. Stern
5
, Leon Raskin
5
, Guo
-
Min Li
1
, David H. Sherman
2,6,*
,
Jacqueline K. Barton
3,*
and Stephen B. Gruber
1,*
Table of Contents
I.
Methods
A
.
Determination of Trans Chromosomal Configuration of
........................
......2
MUTYH Gene
Variants
B.
Identification of APC gene G:C
T:A Transversions
...........................
......2
C.
Cloning of Wild Type and Mutant MUTYH Expression P
lasmids
....................2
D.
Preparation of Protein
...............................................................
.........3
E.
DNA Substrates for Glycosylase and Binding Assays
..............................
....3
F.
DNA Glycosylase Assay
...................................................
.................4
G.
Multiple Turnover assay: Active Site Titration
..............................
............4
H.
Binding Kinetics
(
Biolayer Interferometry)
.................................
............4
I.
Fe Elemental Analysis
.......................................
...............................5
J.
DNA synthesis and
purification for electrochemistry
..............................
....
5
K.
Electrochemistry on DNA self
-
assembled monolayers (SAMs)......
...............6
L
.
EPR spectroscopy
............................................................
...............
.
7
II. Establishment of Trans Chromosomal Configuration (Supplementary Figure 1)
...
8
III.
Demonstration G:C to T:A Transversion (
Supplementary
Figure
2)
............
......9
IV.Size
-
Exclusion, Fast Protein Liquid Chromatography of MUTYH Protein
.........
.
10
(Supplementary Figure 3).
V
.
SDS
-
PAGE of Purified Monomeric MUTYH Proteins (Supplementary Figure 4)
..11
V
I
.
Glycosylase Assay with
Aggregated Protein (
Supplementary
Figure 5
)
............
..12
VII
.
Circular Dichroism Spectra of WT MUTYH and MUTYH C306W
................
.13
(
Supplementary
Figure 6
)
VIII
.
Electrochemical Characterization of MUTYH in HEPES buffer
............
.......14
I
X. HEPES Buffer Electrochemical Characterization
..............................
.........16
(
Supplementary
Figure 7)
X. Abasic Site Discrim
ination by WT MUTYH
(
Supplementary
Figure 8)
.........
.....17
XI
.
Coordinating Cysteine and Neighboring Arginine Residues
...............
............18
Predicted/Reported to occur with MUTYH Associated Polyposis
(
Supplementary
Table 1)
XI
I
.
Somatic Alterations Associated with
Coordinating Cysteine
.....................
.
...
18
and Neighboring Arginine Residues Predicted/Reported to Occur
with
Colorectal and Other Cancers
(Supplementary
Table 2
)
XII
I
. Supplementary References
..........................................
........................
19
Page |
2
I.
Methods
A.
Determination of Trans Chromosomal Configuration of MUTYH Gene Variants
Germline DNA was amplified using the polymerase chain
reaction (PCR) to generate a
935 base pair amplicon that includes the open reading frame positions c.918C>G
(p.C306W) and c.1187G>A (p.G396D). The PCR reaction used the forward primer 5ʹ
-
CCA GGA GAT TTC AAC CAA GC
-
3
ʹ
and the reverse primer 5
ʹ
-
AAG GGT CAA
GGG GTT CAA AT
-
3
ʹ
.
The c.1187G>A mutation creates a unique BglII restriction
endonuclease site which allowed generation of a 719 base pair fragment from the parent
935 base pair amplicon. The shorter 719 base pair fragment was resolved using
agarose
gel electrophoresis, purified and its DNA sequence determined (University of Michigan
(U
-
M) Sequencing Core) to establish the identity of the c.918 position.
B.
Identification of APC gene G:C
T:A Transversions
Tumor DNA was extracted from a formalin
-
fixed, paraffin
-
embedded colonic adenoma
originating from the proband using the RecoverAll Total Nucleic Acid isolation kit
(Ambion). A portion of the mutation cluster region of the
APC
gene
1
was amplified
using PCR with the forward primer 5ʹ
-
TGC CAC AGA TAT TCC
TTC ATC A
-
3ʹ and
the reverse primer 5ʹ
-
CAT GGT TTG TCC AGG
GCT AT
-
3ʹ. The PCR product was
subsequently sequenced (U
-
M Sequencing Core).
C.
Cloning of wild type and Mutant MUTYH
expression plasmids
A plasmid containing the open reading frame for the beta3 isoform of
MUTYH
(NM_001048174.1) was obtained from OriGene (Catalog #: RC201376
-
OR, Rockville,
MD.) and used as a template for the cloning of derivative constructs. Mutant
MUTY
H
construct synthesis was accomplished using PCR
-
based site
-
directed mutagenesis of wild
type
MUTYH
. The wild type and mutants were then cloned as maltose binding protein
(MBP) fusions into pMCSG19
2
between the KpnI and XbaI restriction sites to increase
protein solubility
3
. The plasmids were further modified by
removing the first fourteen
codons encoding the mitochondrial recognition sequence, which alleviated heterologous
protein toxicity. Furthermore, to decrease the capture of truncated heterologous protein,
the N
-
terminus His
6
tag located between the MBP seq
uence and the TEV cleavage site
was removed and a C
-
terminus His
10
tag was attached with a flexible (SG)
7
linker to
increase solvent exposure.
Page |
3
D.
Preparation of Protein
Heterologous MUTYH proteins in
Escherichia coli
strain BL21(DE3) were initially
purified in accordance with previously published protocols using nickel affinity
chromatography with 1 mM DTT
4
. To improve yields and purity, the expression plasmids
were transformed into the
E. coli
expression strain for toxic proteins, BL
-
AI (Invitrogen)
also harboring th
e rare codon plasmid pRARE2
-
CDF
5
. One liter of fresh Terrific Broth
modified with 4% glycerol and 50 μg/mL of antibiotics (ampicillin and streptomycin)
in
a three L baffled flask was inoculated with 25 mL of overnight cultures in the same
medium. Cultures were grown at 37 ºC in a horizontal shaker at 175 rpm until the OD
600
reached approximately 2.5. The temperature was adjusted to 15 ºC, and after 90 mi
nutes,
0.25 mM IPTG and 0.2% arabinose were added. After 12
-
16 hours, cells were harvested
by centrifugation, flash frozen in liquid nitrogen, and stored at
-
80 °C until processing.
Cell pellets were thawed in an ice bath and re
-
suspended in 80 mL of ice c
old 10%
glycerol before the addition of 53 mg/mL of CelLytic Express (Sigma), one tablet of
Protease Inhibitor Cocktail (Sigma) and 20 mM imidazole. The samples were clarified on
a nutator for 30 minutes at 4ºC before the addition of 20 mM of β
-
mercaptoeth
anol. The
crude cell lysate was passed through a 0.45 μm filter in preparation for nickel affinity
chromatography. His
-
tagged proteins were loaded onto a 5 mL HisTrap HP column (GE
Healthcare) at 2 mL/min using an AKTA Explorer FPLC instrument (GE Healthca
re) at 4
ºC. The columns were first washed with 20 column volumes of 93% Buffer A (20 mM
Tris
-
HCl, pH 7.4, 1 M NaCl, 20 mM β
-
mercaptoethanol, 10% glycerol) and 7% Buffer B
(20 mM Tris
-
HCl, pH 7.4, 100 mM NaCl, 500 mM imidazole, 10% glycerol) and the
protei
ns were eluted using a 7
-
100% Buffer B gradient over 10 column volumes.
Fractions containing MBP
-
MUTYH protein (as determined by SDS
-
PAGE), were
pooled. Typical yields of purified protein for MBP
-
MUTYH wild type, Y179C and
G396D were between 10
-
20 mg from
one liter cultures and 0.5
-
1.5 mg of soluble protein
was obtained for MUTYH p.C306W. Monomeric protein was obtained by size exclusion
chromatography using a Superdex 200 10/300 GL column (GE Healthcare) in Buffer C
(20 mM Tris
-
HCl, 100 mM NaCl, 1 mM DTT, 1
0% glycerol). Fractions eluting near the
expected molecular weight (104 kD) were collected, partitioned into aliquots, flash
frozen in liquid nitrogen and stored at
-
80 ºC until further use.
E.
DNA Substrates for Glycosylase and Binding Assays
All oligonucleotides (Integrated DNA Technologies, Coralville, Iowa) were purchased
PAGE purified. Duplexes were obtained by heating 50
l 50 μM complementary strands
at 85 ºC then decreasing the temperature by 0.5 ºC every 30 seconds until attaining room
temperature. The FAM labelled 8
-
oxoG:A duplex used in the DNA glycosylase assay
consisted of 5ʹ
-
ACA AAG AAC TTA TAG CTC CTC CTT GAG CAC ACA GAG GTG
TTC GAT GTA GTT G/A/C GCA GGA CGG GTT CAG T/
6
-
FAM/
-
3ʹ and 3ʹ
-
TGT TTC
TTG AAT ATC GAG GAG GAA CTC GTG TGT CTC
CAC AAG CAT GAT CAA
C/
8oxoG
/G CGT CCT GCC CAA GTC A
-
5ʹ. The biotin labeled 8
-
oxoG:A duplex used
in the binding assay consisted of 5ʹ
-
/BiotinTEG/
AC AAA GAA CTT ATA GCT CCT
CCT TGA GCA CAC AGA GGT GTT CAT GTA GTT G/A/C GCA GGA CGG GTT
CAG T
-
3ʹ and the 8
-
oxo
G oligomer.
Page |
4
F.
DNA Glycosylase Assay
The DNA glycosylase assay was adapted as previously reported
4,6,7
. The activity was
evaluated by providing 10 nM of DNA substra
te containing a single 8
-
oxoG:A mismatch
to wild type or mutant MBP
-
MUTYH proteins (0
-
1000 nM) at 37ºC in a buffer (20 mM
Tris
-
HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.1 mg/mL BSA).
Reactions were quenched after 1 hour with 80 mM NaOH followed
by heating the
samples to 90 ºC for 4 minutes, cooled and then diluted with three volumes of formamide
spiked with GeneScan™ 500 LIZ™ Size Standard (Invitrogen). DNA fragmentation was
determined employing capillary electrophoresis (U
-
M DNA Sequencing Core
, ABI 3730
DNA Analyzer). Traces were analyzed using Peak Scanner
TM
Software (version 1.0,
Applied Biosystems). The percent of excised DNA was calculated as the ratio of the 6
-
FAM peak area migrating at 44 oligonucleotides to the total peak area (at 44 and
66
oligonucleotides).
G.
Multiple Turnover assay: Active Site Titration
The multiple turnover assay was adapted as previously reported
4,6,7
. Reactions were
analyzed for scission of 10 nM FAM
-
labeled 8
-
oxoG duplex DNA after the addition of
MUTYH protein. The total protein concentrations, selected to give a burst amplitude in a
detectible range, were 25, 2670, 500 and 25 nM of MUTYH wild type, Y179C, C306W
and G396D, respectively. Samples were drawn over
a 20 minute time course and
processed as described above. The cleaved product concentration, [P], was fitted with
Equation 1 to determine the amplitude of the burst (A
0
), k
B
(rate constant during the burst
phase) and k
L
(rate constant for the linear phase
).
Equation 1:
[P] = A
0
[1
-
exp(
-
k
B
t)]+k
L
t
The percent of active protein was calculated as a ratio of the A
0
to total protein
concentration.
H.
Binding Kinetics
(
Biolayer Interferometry)
All biolayer interferometry measurements were made on an Octet RED
instrument (Pall
ForteBio, Menlo Park, CA) using streptavidin (SA) biosensors
8
. Assays were performed
in 96
-
well black microplate
s at 25 ºC and 1000 rpm. All volumes were 200 μL. All
proteins were buffer exchanged using PD
-
10 columns (GE Healthcare) pre
-
equilibrated
with PBS then serial diluted (25, 12.5, 6.25, 3.125, 0.78125 nM) into working volumes
with 1X Kinetics Buffer (Pall Fo
rtBio;10 mM Phosphate, pH 7.4, 150 mM NaCl, 0.02%
Tween
-
20, 1 mg/mL BSA). The biotinylated duplex DNA was first immobilized onto the
SA biosensors for 300 seconds and then equilibrated in 1X Kinetics Buffer for 300
seconds. Protein association was performe
d for 150
-
300 seconds followed by dissociation
into 1X Kinetics Buffer for 900 seconds. A reference sensor with immobilized dsDNA
was subtracted from each data set. Shift data was analyzed with ForteBio’s Analysis
software (version 7.1). Kinetic parameters
k
on
and k
off
and affinity (K
D
) were determined
from a
global non
-
linear regression of association and dissociation binding kinetics using
a 1:1 Langmuir binding model.
Page |
5
I.
Fe Elemental Analysis
The presence of elemental Fe within MUTYH protein samples was
determined using a
Thermo Scientific Element2 ICP
-
HRMS
9
. Purified proteins were dial
yzed overnight to
remove glycerol and to allow equilibration with blank buffer (20 mM Tris
-
HCl, pH 7.5,
100 mM NaCl and 1 mM DTT) using a 10K MWCO Slide
-
A
-
Lyzer dialysis cassettes
(Thermo Scientific).
J.
DNA Synthesis and Purification for E
lectrochemistry
Thiol, FA, and OG modified DNA strands were prepared on an automated DNA
synthesizer (Applied Biosystems) and purified by HPLC on a PLRPS column (Agilent)
as described in previously published protocols
10
; unmodified st
rands were ordered from
IDT and purified by HPLC. For electrochemistry, 50 μL 50 μM complementary DNA
strands were degassed and annealed in storage buffer (5 mM sodium phosphate, pH 7.0,
50 mM Na
Cl) in equimolar amounts by a 5
-
minute incubation at 95
o
C
followed by slow
cooling (1.5 hours) to RT on a thermocycler. Well
-
matched (WM) duplex DNA, DNA
with an FA:OG lesion, and substrates containing an abasic site were all prepared in this
way. Duplex sequences were as follows:
WM DNA
5
′
–
ACT GAA CCC GTC CT
G GCT CAA CTA CAT GAA CAC CTC
–
3
′
3
′
–
TGA CTT GGG CAG GAC GCA GTT GAT GTA CTT GTG GAG
–
5
′
–
C6 Thiol
FA:OG DNA
5
′
–
ACT GAA CCC GTC CTG GC
OG
CAA CTA CAT GAA CAC CTC
–
3
′
3
′
–
TGA CTT GGG CAG GAC GC
FA
GTT GAT GTA CTT GTG GAG
–
5
′
–
C6 Thiol
Abasic DNA
5
′
–
ACT GAA CCC GTC CTG GCT CAA CTA CAT GAA C
Ab
C CTC
–
3
′
3
′
–
TGA CTT GGG CAG GAC GCA GTT GAT GTA CTT GTG GAG
–
5
′
–
C6 Thiol
OG = 8
-
oxoguanine, FA = 2ʹ
-
fluoroadenine, Ab = abasic site
Page |
6
K.
Electrochemistry on DNA self
-
assembled monolayers (SAMs)
Electrochemical characterization of MUTYH was carried out on a multiplexed chip
platform consisting of 16 individually
-
addressable gold electrodes separable into four
quadrants
10
. Self
-
assembled DNA monolayers were form
ed by adding 25 μL 25 μM
duplexed DNA in phosphate buffer (5 mM sodium phosphate, pH 7.0, 50 mM NaCl) to
each quadrant of the chip and incubating overnight. After monolayer formation, gaps in
the film were eliminated by backfilling for 45 minutes at RT wit
h 1 mM 6
-
mercapto
-
1
-
hexanol in phosphate buffer with 5% glycerol. The surface was then extensively rinsed in
phosphate buffer, followed by protein storage buffer (described below). To compare
different monolayer morphologies, DNA was incubated in phosphate
buffer as described
to generate low
-
density DNA monolayers (surface coverage of ~15 pmol/cm
2
) or in the
presence of 100 mM MgCl
2
to form high
-
density monolayers (surface coverage of ~40
pmol/cm
2
)
11,12
. Bulk electrolysis experiments were performed using gold rod electrodes
in a custom
-
made electrochemical cell. Experiments were carried out in air unless
otherwise noted; anaerobic experiments were performed in a gl
ove bag (Coy) under a
95% N
2
/5% H
2
atmosphere.
MUTYH concentration was determined by UV
-
vis
ible spectroscopy
, using an extinction
coefficient of 17000 M
-
1
cm
-
1
at 410 nm to determine [4Fe4S] cluster concentration and
102330 M
-
1
cm
-
1
at 280 nm to determine total protein conce
ntration. C
luster loading was
determined by dividing [4Fe4S] cluster concentration by total protein concentration, and
was typically around 15%. Initial characterization was carried out in Tris storage buffer
(20
mM Tris, pH 7.4, 100 mM NaCl, 1 mM DTT, 10% glycerol v/v), while later
electrochemical and spectroscopic experiments used a HEPES buffer (20 mM HEPES,
pH 7.4, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA, 10% glycerol v/v). MUTYH was
transferred into HEPES using Amic
on 10 kDa MW cutoff spin tubes (Millipore
Biomedicals) at 4
o
C.
Once in an appropriate buffer, MUTYH was added to a multiplexed chip and incubated
for several hours with cyclic and square wave voltammetry (CV and SQWV,
respectively) scans taken once per ho
ur. In typical experiments, CV scans were taken in a
potential window of
-
0.188 to 0.412 V vs NHE at a scan rate of 100 mV/s, while SQWV
scans were taken at a frequency of 15 Hz with 0.025 V amplitude. To plot the scan rate
dependence of CV current, scans
in the same window were carried out at 20, 50, 80, 100,
200, 500, 750, and 1000 mV/s. All experiments were performed on a CH Instruments
potentiostat with an Ag/AgCl reference in 3 M NaCl and Pt wire counter electrode.
Potentials were converted to NHE by a
dding 212 mV to the measured potentials,
accounting for both the salt concentration (209 mV according to BASi®) and ambient
temperature
13
. Bulk electrolysis was carried out at 0.412 mV versus NHE, and yields
were estimated by subtracting the total charge passed with only buffer present from that
passed when MUTYH was included. All buffer components were purchased from Sigma
-
Ald
rich, the Ag/AgCl reference electrode was purchased from BASi®, and the Pt wire
counter electrode was purchased from the Kurt J. Lesker Company.
Page |
7
L.
EPR spectroscopy
Continuous wave X
-
band EPR was carried out at 10 K on a Bruker EMX instrument.
Samples
were prepared aerobically, using 150 μL 5
–
15 μM MUTYH in parallel with a
storage buffer blank. Spectra were taken from the summation of 9 sweeps at 12.88 mW
microwave power, 2 G modulation amplitude, and a receiver gain of 5.02 x10
3
.
Page |
8
II.
Establishment of Trans Chromosomal Configuration (Supplementary Figure 1)
Supplementary Figure 1.
The
c.918C>G MUTYH variant is situated trans relative to the
c.1187G>A MUTYH mutation.
The c.1187G>A (p.G396D)
MUTYH
mutation creates a
unique
BglII restriction enzyme site which we employed to isolate the chromosomal DNA strand
containing the c.1187G>A
MUTYH
mutation. A 957 bp fragment encompassing the
MUTYH
open reading frame nucleotide positions 918 and 1187 was generated with PCR and then
restriction enzyme digested with BglII. The digestion products were resolved on 1.5% agarose
gel and the lower molecular weight DNA band isolated (arrow). Sequencing of
the lower
molecular weight band demonstrated the wildtype cytosine nucleotide at position 918 consistent
with a trans configuration of the c.1187G>A and c.918C>G
MUTYH
alterations.