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
,
Janice Ortega
5
,
Ralph H. Stern
6
,
Leon Raskin
7
,
GuoMin 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
Abstract
The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA.
Homozygous
MUTYH
mutations underlie the autosomal, recessive cancer syndrome
MUTYH
associated polyposis. We report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan
residue in place of native cysteine, that ligates the [4Fe4S] cluster in a patient with colonic
polyposis and family history of earlyage colon cancer. In bacterial MutY, the [4Fe4S] cluster is
redox active, allowing rapid localization to target lesions by longrange, DNAmediated signalling.
In the current study, using DNA electrochemistry, we determine that wildtype MUTYH is
similarly redoxactive, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to
Correspondence to: David H. Sherman; Jacqueline K. Barton; Stephen B. Gruber.
These authors wish it to be known that, in their opinion, the first 3 authors should be regarded as joint First Authors: Kevin J.
McDonnell, Joseph A. Chemler, Phillip L. Bartels.
Publisher’s note:
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Supplementary information is available for this paper at
https://doi.org/10.1038/s415570180068x
.
Author contributions
K.M., J.C., P.B., E.O., D.S., J.B. and S.G. conceived and designed the experiments. K.M., J.C. and P.B. cowrote the paper with input
from all authors. R.S., L.R., M.M., J.O. and G.L. contributed materials and analysis tools. K.M., J.C. and P.B. performed the
experiments.
Competing interests
The authors declare no competing interests.
HHS Public Access
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[3Fe4S]
+
, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to
decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic
DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the
essential role of redox signalling by the [4Fe4S] cluster.
Graphical abstract
The [4Fe4S]
2+
clustercontaining DNArepair enzyme MUTYH helps safeguard the integrity of
Watson–Crick base pairing and the human genetic code. The MUTYH [4Fe4S]
2+
cluster mediates
DNA redox signalling and DNA lesion identification. Now, a MUTYH pathologic variant
associated with catastrophic [4Fe4S]
2+
cluster redox degradation, impairment of DNA signalling
and human colonic tumorigenesis has been identified.
In cells sustaining oxidative damage, genomic guanine residues are frequently oxidized to 8-
oxo7,8dihydroguanine (8oxoG). Unlike guanine, 8oxoG can pair effectively with either
cytosine or adenine bases, with potentially serious mutagenic consequences[
1
]. A DNA
glycosylase conserved among species from bacteria to humans, known in humans as
MUTYH, removes adenine from these 8oxoG:A mispairs as part of the base excision repair
pathway. In humans, germline
MUTYH
mutations that impair enzymatic activity lead to an
increase in G:C>T:A transversions that have been shown to result in missense mutations in
the
APC
tumour suppressor gene in epithelial cells lining the colon. Mutations in
APC
are
the first recognizable genetic events that initiate malignant transformation of normal colonic
epithelia into polyps, specifically adenomas, before the acquisition of other mutations that
complete the neoplastic conversion sequence from normal tissue through adenoma to
carcinoma[
2
]. Biallelic mutations of
MUTYH
give rise to the autosomal recessive cancer
genetic syndrome,
MUTYH
associated polyposis (MAP)[
3
]. Typically, by their fifth decade,
MAP patients develop 10–100 colonic polyps[
4
].
MUTYH
variants are common, with a
prevalence of at least 1–2% among western Europeans[
5
], and the colorectal cancer risk
increases nearly 2 and 100fold for mono and biallelic
MUTYH
mutations, respectively[
6
].
The MUTYH protein comprises three major regions[
7
]: the N terminus, which contains the
endonuclease III sixhelix barrel catalytic domain, the interdomain connector (IDC) and the
C terminus, corresponding to protein residues 1–306, 315–366 and 368–500, respectively.
The MUTYH N terminus contains a [4Fe4S]
2+
cluster ligated by four cysteine residues
;
although metabolically expensive, the cluster is conserved in MutY homologues across all
domains of life[
8
]. A rare exception is the yeast endonuclease III homologue Ntg1, which
has no cluster, but one is present in a second yeast homologue (Ntg2)[
9
]. Indeed, the only
organisms lacking a cluster in any MutY homologue are specialized anaerobes subject to
lower levels of oxidative stress[
10
]. Studies performed on
Escherichia coli
MutY and its
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homologue endonuclease III (EndoIII) have demonstrated that the cluster is unnecessary for
structural integrity and is largely redoxinert in solution[
11
,
12
]. However, when
E. coli
MutY and EndoIII were incubated on duplex DNAmodified gold electrodes, a reversible
redox signal centred near 80 mV versus normal hydrogen electrode (NHE) was observed for
both proteins, and was identified as the [4Fe4S]
3+/2+
couple, an assignment supported by
electron paramagnetic resonance (EPR) spectroscopy[
13
]. Subsequent experiments with
EndoIII on a graphite electrode in the presence and absence of DNA revealed that binding to
the negatively charged DNA backbone shifts the redox potential of these proteins by about
−200 mV, activating the cluster towards oxidation and resulting in a significant increase in
binding affinity of the oxidized [4Fe4S]
3+
form of the enzyme relative to the native
[4Fe4S]
2+
form[
14
].
These studies have led to a model in which [4Fe4S] base excision repair (BER) proteins with
similar DNAbound redox potentials use reversible redox exchanges to signal to one another
across the genome, taking advantage of the unique ability of DNA to conduct charge across
the
π
stacked base pairs (bps) in a process known as DNAmediated charge transport (DNA
CT)[
15
,
16
]. DNA CT has both a very shallow distance dependence and an exquisite
sensitivity to even slight disruptions in base pair stacking, making it an ideal lesion reporter.
In our CT signalling model, oxidative stress generates highly reactive species, such as
guanine radicals, which can then oxidize proteins including MutY[
17
]. If another [4Fe4S]
protein is bound at a distal site and the intervening DNA is undamaged, it can send an
electron through the DNA to reduce the first protein. Following reduction, the protein’s
affinity for DNA is decreased and the protein dissociates to another region of the genome,
while the oxidized protein remains bound. In the presence of a lesion, DNA CT is impaired,
and the oxidized protein will remain bound and diffuse towards the site of damage. Thus,
DNA CT constitutes a means for [4Fe4S] proteins to scan a vast genome on a relevant
timescale and redistribute in the vicinity of lesions. In the case of
E. coli
, longrange
signalling by DNA CT has been estimated to reduce the damage search time from 45 min to
10 min or less[
18
].
In the present study, we describe a novel MUTYH variant, p.C306W, identified in a patient
exhibiting colonic polyposis. Using an
E. coli
overexpression system, this variant was
generated together with wildtype (WT) MUTYH and the wellcharacterized pathologic
variants Y179C and G396D. We used electrochemistry, UV–vis and EPR spectroscopy to
compare the redox properties of these four MUTYH variants. Enzymatic activity and DNA
binding parameters were determined using glycosylase assays and biolayer interferometry
(BLI), respectively. Together, these results provide strong evidence for a primary function of
the [4Fe4S] cluster in DNAmediated redox signalling and establish MUTYH C306W as a
pathogenic variant, enhancing our understanding of the role of the [4Fe4S] cluster in human
disease.
Results
Identification and functional deficiencies of a novel MUTYH variant
A novel germline
MUTYH
variant, c.918C>G (p.C306W), together with the previously well-
described
MUTYH
mutation c.1187G>A (p.G396D), were identified in a patient with
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colonic polyposis whose family history was significant for earlyage colon cancer. The
cysteine at position 306 represents one of the four cysteine residues that mediate integration
of the conserved MUTYH [4Fe4S] cluster. In bacterial MutY, cluster loss is associated with
decreased protein function[
12
], which suggests that in MUTYH the C306W variant may
affect the integrity of the [4Fe4S] cluster and represent a pathologic mutant (Fig. 1a
illustrates the structure of bacterial MutY and identifies corresponding residues in MUTYH).
Supporting the potential pathogenicity of the novel ~918C>G variant, we established that
this variant is situated in a trans chromosomal configuration relative to the ~1187G>A
MUTYH
mutation (Supplementary Fig. 1). Further evidence of the pathogenic nature of the
~918C>G variant was apparent in the sequence of the
APC
gene in somatic DNA originating
from a colonic adenoma from the patient. This sequencing revealed the presence of a
G:C>T:A transversion in the
APC
gene, which is the hallmark genetic lesion indicative of
deficient
MUTYH
mediated DNA enzymatic repair[
2
] (Supplementary Fig. 2).
Four MUTYH proteins (WT, the wellcharacterized mutants G396D and Y179C, and the
novel variant C306W) were overexpressed in
E. coli
and purified by nickel affinity
chromatography. Monomeric MUTYH proteins were produced by treatment with 20 mM
β‐
mercaptoethanol, followed by size exclusion fast protein liquid chromatography with
validation by UV spectroscopy (Supplementary Figs. 3 and 4). Relative to WT MUTYH,
G396D MUTYH demonstrated a significant decrease in glycosylase activity and Y179C a
severe deficiency
;
comparative assessment of C306W MUTYH revealed a virtual absence of
activity, thus establishing its pathogenicity (Fig. 1b). The glycosylase activities of
aggregated MUTYH proteins were also assessed
;
in these experiments C306W was
similarly deficient in glycosylase activity (Supplementary Fig. 5).
For a more complete functional comparison of the four MUTYH variants, we conducted
timecourse glycosylase assays under multiple turnover conditions in order to quantitatively
determine the proportion of active enzyme in each sample[
19
,
20
,
21
]. These assays were
performed using dsDNA containing an 8oxoG:A mispair together with varying
concentrations of MUTYH proteins. The experimental results demonstrate an initial burst of
adenine excision activity proportional to the active fraction,
A
o
, of the protein sample (Fig.
1c and Table 1). The excision reaction rate constants,
k
B
and
k
L
, were determined for the
exponential and linear phases of the reaction, respectively (Table 1). Both WT MUTYH and
G396D proteins had comparable linear rates for turnover and the highest fraction of active
protein. In contrast, the C306W MUTYH mutant was essentially devoid of adenine excision
activity and Y179C had no detectable turnover. The fractions of active MUTYH were then
used to correct for the total amount of protein used in the glycosylase assay (Fig. 2d),
confirming that WT MUTYH and G396D mutant had comparable activities, while C306W
and Y179C mutants displayed poor activity. The poor activity observed in MUTYH C306W
could have two possible explanations: either that this mutant was catalytically inactive or it
was unable to bind specifically to DNA (as is the case with low activity in the weakly bound
Y179C).
To help distinguish between these possibilities, we used BLI[
22
] to measure the binding
parameters of the MUTYH proteins. We compared the binding of WT MUTYH and the
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G396D, Y179C and C306W mutants to DNA containing an 8oxoG:A mispaired duplex.
Table 1 summarizes the binding kinetics. Relative to WT MUTYH, the G396D and Y179C
variants demonstrated increasing values of
K
D
primarily due to decreased association rates.
There was no detectable binding for the C306W mutant within the protein concentration
range tested, suggesting that the low activity levels observed in this mutant were due
primarily to ineffective DNA binding.
Together, these data demonstrate the functional deficiency and establish the pathogenicity of
the C306W MUTYH variant. In bacterial MutY, [4Fe4S] cluster loss is associated with
decreased protein function[
12
], which suggests that in MUTYH the C306W variant may
affect the integrity of the [4Fe4S] cluster, accounting for the pathogenicity of this mutant.
To assess the integrity of the [4Fe4S] cluster, iron loading of the clusters of WT MUTYH
and mutants Y179C, G396D and C306W were compared by quantifying the iron present in
each sample using inductively coupled plasma–highresolution mass spectrometry (ICP-
HRMS) for elemental analysis[
23
]. Consistent with disruption of the Fe–S cluster loop in
the C306W variant, this protein exhibited substantially lower iron content relative to the
other MUTYH proteins tested (Table 2). However, UV–vis spectra taken from disrupted
aggregates of all four variants distinctly showed the broad peak centred at 410 nm that is
characteristic of a [4Fe4S] cluster, indicating that MUTYH C306W is still capable of
binding an intact cluster and further suggesting that loading by cellular machinery is still
effective (Supplementary Fig. 6). In addition, circular dichroism (CD) spectra of WT
MUTYH and the C306W mutant were indistinguishable, confirming that no global
conformational changes were induced by this mutation (Supplementary Fig. 6). Thus, the
low cluster content, as measured by ICPHRMS, was instead tentatively associated with
decreased protein stability in this mutant, and subsequent electrochemical and EPR
experiments were used to more reliably examine the [4Fe4S] cluster properties in detail.
DNA-bound electrochemistry of WT and mutant MUTYH
Having observed that MUTYH C306W appeared to incorporate less iron despite its capacity
to bind an intact cluster, we next assessed its redox properties on DNAmodified gold
electrodes alongside WT, G396D and Y179C MUTYH (Fig. 2). We reasoned that
electrochemical analysis would aid these studies for two reasons: first, access to highly
purified WT MUTYH allowed us to determine if the human protein behaved in a manner
similar to its bacterial counterpart, and, second, electrochemical monitoring would provide
an effective way to assess the stability of MUTYH C306W over time. Specifically, we
expected that the predicted instability of MUTYH C306W would result in electrochemical
signals that were either smaller than WT or less stable over time.
In these experiments, MUTYH was incubated in storage buffer (20 mM Tris, pH 7.4, 100 mM
NaCl, 1 mM DTT, 10% glycerol vol/vol) and periodically scanned by cyclic voltammetry
(CV) and square wave voltammetry (SQWV). Aggregated protein preparations were treated
with 20 mM
β
mercaptoethanol and exchanged into storage buffer with fresh DTT
immediately before electrochemical analysis. UV–vis spectroscopy confirmed that treated
proteins were monomeric and contained intact [4Fe4S] clusters. Notably, cluster loading in
monomeric WT MUTYH prepared from disassembled aggregates was significantly lower
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than expected (~15% as determined from the
A
410
:
A
280
ratio), but the low loading actually
turned out to be advantageous for these experiments. Because apoproteins may bind some
available DNA on the surface and thus decrease signal amplitude, the most important factor
in making direct comparisons between different MUTYH proteins on an electrode is not
absolute loading (although high levels are ideal) but that each variant is similarly loaded
with a [4Fe4S] cluster. Fortunately, this turned out to be the case, with the MUTYH C306W
samples also ~15% loaded as determined by UV–vis spectroscopy (Supplementary Fig. 6).
To test multiple conditions simultaneously, DNA monolayers were prepared on multiplexed
gold electrodes, which enabled up to four experiments to be conducted in parallel[
24
]. In
this case, half of the available quadrants consisted of unmodified well matched (WM) DNA
and half of substrate trap FA:OG DNA, which was included in an effort to enhance the
signal amplitude by increasing the DNA binding affinity. In addition, both highdensity
(formed in the presence of 100 mM MgCl
2
) and lowdensity monolayers were compared on a
single chip. DNA surface density is an important parameter for protein experiments[
24
]:
highdensity films have more DNA on the surface (30–50 pmol/ cm
−2
), which can improve
DNAmediated signalling by sterically hindering large proteins, while lowdensity films
contain less DNA (15–20 pmol cm
−2
), which can be more readily accessible to proteins[
25
,
26
]. Overall, the effect of each type of film is likely to depend strongly on the particular
protein being studied, and both have been used in previous studies[
24
].
In the case of WT MUTYH at a concentration of ~2.5 μM [4Fe4S] cluster, a reversible redox
signal with a midpoint potential of 106 ± 1 mV versus NHE was immediately apparent, and
increased over the course of the experiment (Fig. 3). The midpoint potential was similar to
the 65–95 mV (versus NHE) range reported for other [4Fe4S] proteins bound to DNA, with
the slightly higher potential of MUTYH most probably attributable to the distinct buffer
conditions. Although the signals were relatively small, they were readily quantifiable: on
lowdensity films containing WM DNA, CV peak areas were 31 ± 1 × 10
−2
nC for the reductive
peak and −33 ± 2 × 10
−2
nC for the oxidative peak, while the equivalent values on highdensity
films were 25 ± 2 × 10
−2
nC and −27 ± 3 × 10
−2
nC, respectively. Interestingly, no significant
differences in signal intensity were observed between WM and FA:OG DNA, with CV
reductive and oxidative peak charges of 28 ± 1 × 10
−2
nC and −34 ± 4 × 10
−2
nC on lowdensity
FA:OG DNA films and 24 ± 2 × 10
−2
nC and −24 ± 1 × 10
−2
nC on highdensity films. The
FA:OG substrate trap is known to increase the binding affinity of the very similar murine
Mutyh on a 30mer duplex by an order of magnitude[
27
], and the absence of any clear
change in signal intensity suggests that our system is not sufficiently sensitive to detect this
difference. Several possible explanations exist for this insensitivity. First, it may be that the
absolute amount of accessible DNA on either surface is too low to detect a difference
between WM and FA:OG substrates. Alternatively, the significant amount of apoprotein
present may have blocked some of the accessible DNA from fully loaded protein. A further
complicating factor may be the presence of DNA tethered to a surface rather than in
solution, which might lower the chances of protein encounter. Despite these potential
limitations, we were able to definitively observe and quantify signals from DNAbound
MUTYH.
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The signal from MUTYH C306W (also at the ~2.5 μM [4Fe4S] cluster) was comparable in
both potential and maximum size to WT, although the signal size was considerably more
variable between experiments. Although it was not clearly CTdeficient, as might have been
expected, the C306W signal decreased in size at a steady rate after 1–2 h of incubation,
consistent with the loss of iron seen by ICPHRMS. In addition, the intensity of a second,
irreversible peak centred around −50 mV versus NHE increased as the reversible
[4Fe4S]
3+/2+
signal decayed. This secondary peak was unprecedented among BER proteins
on DNAmodified electrodes, but its growth in parallel with loss of the reversible signal
suggested that it was some form of degradation product.
Although all of the DNAprocessing enzymes studied thus far have shown stabilization of the
[4Fe4S]
3+
form on DNA binding to yield a reversible [4Fe4S]
3+/2+
signal on an electrode,
loss of iron by the oxidized [4Fe4S]
3+
species to form the [3Fe4S]
+
cluster has been
reported in bacterial MutY and EndoIII when the samples were frozen for EPR under
aerobic conditions[
13
]. As this is the first step in cluster degradation, we considered the
[3Fe4S] cluster to be a likely candidate for the identity of this unexpected MUTYH C306W
species. At approximately −50 mV versus NHE, the MUTYH C306W secondary peak fell
within the range of reported [3Fe4S]
+
cluster potentials[
28
], supporting assignment to this
species. The irreversible nature of the signal was unusual, given that [3Fe4S] clusters can
typically access a reversible 1
+
/0 redox couple, but, given the significant effect of even a
single unit of cluster charge on DNA binding affinity[
14
], irreversibility could be explained
as protein dissociation from DNA on reduction to the neutral [3Fe4S]
0
cluster.
Confirming that the [4Fe4S] cluster degradation observed in MUTYH C306W was unique to
this mutant, electrochemical analysis of DNAbound Y179C and G396D (both 2.5 μM in
storage buffer) yielded reversible signals at nearly the same potential as WT with no
secondary peak present (Table 3). Like WT, the signals from both of these variants increased
over time and remained stable for several hours (Fig. 3). Notably, the Y179C signal was only
about half as large as that for the WT, which is consistent with the reported lower binding
affinity of this mutant relative to both WT and G396D (7.5 nM for Y179C versus 2.2 and
4.9 nM for WT and G396D, respectively)[
19
]. Overall, however, both mutants were more
similar to WT than C306W in their redox properties, which was unsurprising given that the
cluster in these variants is unaltered. The propensity for the MUTYH C306W cluster to
degrade by redox activity provides a possible explanation for the low DNAbinding affinity
observed with BLI binding studies, as previous MutY studies have demonstrated that
apoprotein lacking cluster remains structurally intact, but exhibits defective DNA
binding[
12
].
Characterization of the C306W degradation product
In an effort to characterize the MUTYH C306W cluster degradation product more fully, we
proceeded to assess its dependence on oxygen, which is often involved in [3Fe4S]
+
cluster
formation[
13
]. Specifically, we employed electrochemical and UV–vis spectroscopic
analysis to compare aerobically oxidized proteins with those maintained in an anaerobic
environment. For an effective comparison, a single C306W sample was concentrated and
one half of the sample was diluted to 2.5 μM in degassed buffer and placed on a chip
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containing lowdensity WM DNA in an anaerobic glove bag (95% N
2
/5% H
2
atmosphere),
while the other half was maintained in aerobic conditions and oxidized on a DNAmodified
gold rod electrode held at 0.412 V versus NHE. The anaerobic sample was scanned
periodically by CV and SQWV, and, following electrolysis, the oxidized sample was
transferred to the glove bag and added to a separate quadrant on the same chip. In addition to
electrochemistry, UV–vis spectra were recorded before and after electrolysis both to observe
changes in the 410 nm peak and to ensure that the oxidized sample did not aggregate.
Quantification of the total charge passed during electrolysis indicated nearcomplete
oxidation of the aerobic protein by ~60 min. Before electrolysis, the UV–vis spectrum
showed the broad peak centered at 410 nm characteristic of a [4Fe4S] cluster, but, after
oxidation, the absorbance increased over a broad range from 700 to 300 nm, with a poorly
defined peak around 410 nm and a substantial shoulder between 400 and 300 nm (Fig. 3).
Such absorbance features are a general characteristic of cluster oxidation, although UV–vis
spectra alone are insufficient to precisely identify the oxidized species generated[
29
].
Importantly, the 280 nm peak remained sharp and distinct even after oxidation, and the
spectrum was not elevated at 800 nm, demonstrating that the protein had not aggregated and
confirming that all changes were due solely to cluster oxidation. This result stands in stark
contrast to the soluble MUTYH aggregates observed in both WT and C306W in the absence
of DTT, which were visibly cloudy with a Ushaped UV–vis spectrum characteristic of
aggregation, highly elevated absorbance at 800 nm, no distinct [4Fe4S] peak, and a very
slight 280 nm peak visible only as a shoulder (Supplementary Fig. 6).
CV of the aerobically oxidized C306W MUTYH variant revealed an irreversible peak
comparable in size to the main reversible peaks
;
in contrast, the equivalent peak in the
anaerobic sample was much smaller than the main peak and had not changed from initial
levels (Fig. 3). Furthermore, the reversible signal of the anaerobic sample increased over
time and exceeded even the strongest signals observed for aerobically incubated WT
MUTYH, which was even more intriguing given that the anaerobic sample had been
incubating on the electrode for several hours. Supporting greater instability of the C306W
[4Fe4S] cluster, aerobic oxidation of WT MUTYH gave low bulk electrolysis yields, and no
readily apparent irreversible peak was present by CV. In addition, the UV–vis spectra of WT
before and after oxidation were indistinguishable. Taken together, the apparent sensitivity of
MUTYH C306W to oxidation and degradation in air along with the absence of any
observable degradation in aerobically oxidized WT MUTYH supported the assignment of
the secondary peak to a [3Fe4S]
+
cluster, although these techniques alone could not verify
this identity.
EPR spectroscopy of MUTYH
Having confirmed that the C306W degradation product was an oxidized species forming
under aerobic conditions, we turned to EPR spectroscopy as a final step towards its
definitive identification. EPR provides a means of distinguishing among different
paramagnetic species and is commonly used to study Fe–S proteins[
28
,
29
,
30
]. Although
EPR analysis can be very informative, there were two general concerns with respect to
MUTYH. First, EPR experiments are generally performed with significantly higher levels of
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concentrated protein than those used in our electrochemical experiments with MUTYH:
signals have been reported for 10 μM
E. coli
EndoIII[
13
] and ~9 μM DNA polymerase
δ
(ref.
[
31
]), but even these were still three to four times more concentrated than the MUTYH
samples. Second, the low temperatures necessary to resolve signals from [4Fe4S] clusters
(10–35 K) require the samples to be frozen before analysis, which can impact protein
stability if the buffer pH changes with temperature, as is the case for the Tris buffers used in
our MUTYH studies[
32
].
Therefore, before attempting EPR, all MUTYH variants were concentrated and exchanged
into a HEPES buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 1 mM DTT, 10% glycerol vol/
vol, pH 7.4), and potassium was used in place of sodium as a further precaution to stabilize
the pH at low temperature[
33
]. UV–vis spectroscopy and electrochemistry (Supplementary
Fig. 6
;
for details see Supplementary Section VIII) were used to confirm protein stability in
HEPES and to verify that the redox properties remained comparable. As observed in the
UV–vis spectra (Fig. 3), all MUTYH variants maintained their monomeric form in this
buffer, although they could not be concentrated beyond 15 μM without forming soluble
aggregates. When such aggregates did form, they were readily resolved by simple dilution,
and the UV–vis spectra of WT MUTYH and mutants Y179C and G396D all retained a sharp
410 nm peak with ~15% cluster loading even after an additional freeze–thaw cycle one week
after buffer exchange (Fig. 3). In contrast, the spectrum of the MUTYH C306W protein
closely resembled the aerobically oxidized sample described previously (Fig. 3). Oxidation
may have occurred over the extended aerobic buffer exchange process or during the freeze–
thaw cycle on the day of EPR experiments. Because the extinction coefficient at 410 nm was
unknown for the MUTYH C306W product, we estimated cluster loading in this mutant by
comparing the magnitude of absorbance with the earlier aerobically oxidized sample,
yielding a concentration comparable to the other variants.
All EPR spectra of MUTYH proteins were obtained with 15 μM WT and C306W and 5 μM
G396D and Y179C (the latter mutants were not available in larger amounts). From the
broadened UV–vis absorption, we predicted that the corresponding EPR spectrum would
show evidence of either the [3Fe4S]
+
cluster or a more advanced degradation product, while
WT, Y179C and G396D were expected to be diamagnetic and thus EPR silent.
Unexpectedly, all of the samples showed a small, broad signal with a shoulder at
g
= 2.04
(Fig. 3) most likely attributable to oxidation during the aerobic freezing process, as reported
previously for
E. coli
EndoIII and MutY. Nonetheless, MUTYH C306W protein displayed a
much sharper signal with a clear peak centred at
g
= 2.018 (Fig. 3), which is characteristic of
[3Fe4S]
+
clusters[
28
,
30
]. Importantly, the C306W EPR signal, but not the broad signals of
WT and the other MUTYH variants, closely resembled spectra from chemically oxidized
E.
coli
EndoIII and MutY that were also assigned to the [3Fe4S]
+
cluster[
13
]. Although some
of the C306W [4Fe4S] degradation may have occurred during sample freezing, the
significantly larger and sharper EPR signal relative to the other variants, coupled with the
UV–vis spectrum indicating previous oxidation, contradict this notion.
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Discussion
In the present study we describe a novel MUTYH variant, C306W, and its association with
the development of colonic polyposis and a family history of colon cancer. We determined
that the C306W variant lacks DNA abscission activity and has decreased ability to bind
target DNA, establishing the pathogenicity of this variant. In C306W MUTYH there is loss
of a cysteine residue that ligates the MUTYH [4Fe4S] cluster. This finding raised the
possibility that the loss of the cysteine might disrupt the integrity of the [4Fe4S] cluster and
provide an explanation for the pathogenicity associated with MUTYH C306W. The
observation by ICPHRMS that the [4Fe4S] cluster of C306W exhibits significantly lower
iron content further bolstered this hypothesis and prompted an electrochemical, mechanistic
investigation of the MUTYH [4Fe4S] cluster and the effect of cysteine loss in the C306W
variant.
Towards this end, we present direct evidence of redox signalling in eukaryotic MUTYH.
DNAmodified electrochemical analysis revealed the redox potentials of all MUTYH variants
studied to be in general agreement with earlier work (Table 3), with the potentials as
measured in HEPES buffer almost identical to those obtained for
E. coli
MutY in phosphate
buffer[
13
]. The similarity of the WT MUTYH electrochemical signals to those of the
E. coli
protein strongly supports the notion that the primary function of the conserved [4Fe4S]
cluster is redox activity in all organisms. Furthermore, the DNAmediated nature of this
signal in MUTYH suggests that a process akin to the DNAmediated redoxbased damage
search observed in bacteria may also be present and operating in humans.
Unlike WT MUTYH, the C306W mutant showed an unexpected, and irreversible, reduction
between −50 and −100 mV versus NHE, in combination with loss of the reversible signal at
~100 mV versus NHE
;
EPR spectroscopy confirmed this additional signal to be the [3Fe4S]
+/0
couple. The observed degradation and poor DNA binding in MUTYH C306W are
consistent with the higher DNA binding affinity associated with increasing charge in
[4Fe4S] clusters, in which coulombic effects cause the [4Fe4S]
3+
cluster to bind the DNA
polyanion significantly more tightly than the [4Fe4S]
2+
form[
14
]. The relationship of cluster
charge to binding affinity predicts that the [3Fe4S]
+
and [3Fe4S]
0
degradation products, with
one and zero net charges, would bind much more weakly to DNA than the [4Fe4S]
2+
form,
consistent with the irreversible reduction observed in degraded MUTYH C306W. Overall,
our results suggest that, for MUTYH C306W, ordinary redox activity on DNA would lead to
oxidation to the [4Fe4S]
3+
state, as is typical in these proteins, but the lower stability of the
cluster would promote the loss of an iron atom and irreversible dissociation following a
second redox signalling cycle. Ultimately, this process could result in the low iron content
measured by ICPHRMS, an effect that might well be exacerbated if the dissociated [3Fe4S]
0
form degraded further when removed from the protective environment of DNA. Cluster
degradation in MUTYH C306W is also consistent with the low levels of glycosylase activity
and poor DNA binding affinity as measured by BLI (Table 1), which are attributes of
bacterial MutY following cluster removal[
12
]. This inherent instability of the C306W
[4Fe4S] cluster and consequent loss of function we propose to be a source of pathogenicity
in this MUTYH variant.
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With regard to other potential sources of pathogenicity, we recognize that MUTYH is also
regulated by posttranslational modifications, including phosphorylation and ubiquitination,
that could be altered by this mutation[
34
,
35
]. However, these sites are in different regions of
the protein relative to the [4Fe4S] domain, and are thus unlikely to be affected by this
particular mutation. Thus, redoxstimulated cluster degradation is most likely the primary
cause of pathogenicity in MUTYH C306W.
The irreversible [3Fe4S] cluster signal seen in MUTYH C306W has not been observed
previously in electrochemical studies of DNAprocessing [4Fe4S] proteins, but the signal was
within the same redox potential range reported for the [3Fe4S]
+/0
couple of bacterial Ni–Fe
hydrogenase and fumarate reductase enzymes[
36
,
37
]. In
E. coli
MutY, [4Fe4S] cluster
ligand substitution of the corresponding cysteine residue has been shown to be defective in
DNA binding, similar to the situation with MUTYH C306W[
38
]. We note, however, that
none of the substitutions involving
E. coli
MutY occurred with a residue as bulky as
tryptophan[
38
].
Given the results obtained for MUTYH C306W, it appears probable that mutations in other
residues that alter the region around the [4Fe4S] cluster will be similarly deficient in their
ability to mediate repair of oxidatively damaged DNA
in vivo
[
39
,
40
,
41
,
42
,
43
]. Indeed,
both germline and somatic alterations in other cysteines comprising the [4Fe4S] cluster and
the four arginines that participate in hydrogen bonding to the cysteines coordinating the
cluster[
44
] (Supplementary Tables 1 and 2) have been identified and are associated with
colorectal as well as other cancers[
7
]. It is probable that these mutations also result in
instability, degradation and dysfunction of the [4Fe4S] cluster secondary to the same
mechanisms detailed above. The effects of these lesions as well as the C306W variant
underscore the importance of the [4Fe4S] cofactor in establishing competent MUTYH-
mediated DNA repair.
The current study advances our basic electrochemical understanding of the redox chemistry,
function and integrity of the [4Fe4S] cluster, as well as providing insight into the pathologic
sequelae resulting from disruption of the cluster. Specifically, we have documented and
provided an explanation for a novel mechanism of colonic polyposis and cancer
predisposition linked to electrochemical compromise of the MUTYH [4Fe4S] cluster. Future
studies, we anticipate, will provide additional clarification of the central role of the [4Fe4S]
cluster in MUTYHmediated DNA repair and its underlying electrochemistry.
Reporting Summary
Further information on experimental design is available in the Nature Research Reporting
Summary linked to this article.
Data availability
The data sets generated during and/or analysed during the current study are available from
the corresponding author.
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Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank T. Huston of the W.M. Keck Lab in the Department of Earth & Environmental Sciences at the
University of Michigan for ICPHRMS analyses. This work was funded in part by a Ruth L. Kirschstein National
Research Service Award (GM095065 to J.A.C.), a National Institutes of Health (NIH) grant (R35 GM118101) and
an H.W. Vahlteich Professorship (to D.H.S.), a Ruth L. Kirschstein National Research Service Award and American
Society of Clinical Oncology Young Investigator Award (to K.M.), grant 1R01CA197350 (to S.B.G.), a USC Norris
Comprehensive Cancer Center Support Grant (CA014089 to S.B.G.), an award from the Ming Hsieh Institute for
Engineering—Medicine for Cancer, and support from Daniel and Maryann Fong and the Anton B. Burg Foundation
(to S.B.G.). P.L.B., E.O.B. and J.K.B. acknowledge the NIH (GM126904 to J.K.B.) and Moore Foundation for
financial support. E.O.B. acknowledges NIH training grant T32GM07616 and a Ralph Parsons Fellowship for
support.
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