An Msh3 ATPase domain mutation has no effect on MMR function
Edwards BMC Res Notes
An Msh3 ATPase domain mutation has no effect on MMR function
Yasmin Edwards 0
0 Bronx Community College , 2155 University Avenue, Bronx, NY 10453 , USA
Objective: To demonstrate that the Msh3 ATPase domain is required for DNA mismatch repair and tumor suppression in a murine model. Results: The DNA mismatch repair proteins are members of the ABC family of ATPases. ATP binding and hydrolysis regulates their mismatch repair function. In the current study, a mouse model was generated harboring a glycine to aspartic acid residue change in the Walker A motif of the ATPase domain of Msh3. Impaired ATP mediated release of the Msh2-Msh3GD/GD complex from it's DNA substrate in vitro confirmed the presence of an ATPase defect. However, the mismatch repair function of the protein was not significantly affected. Therefore, mutation of a critical residue within the ATPase domain of Msh3 did not preclude mismatch repair at the genomic sequences tested. Indicating that Msh3 mediated mismatch function is retained the absence of a functional ATPase domain.
DNA mismatch repair (MMR) proteins target and
mediate repair of DNA polymerase errors of replication and
signal the DNA damage response [
]. The MMR system
consists of the highly conserved MutS and MutL
homologues. In eukaryotes, the MutS homologues are Msh2,
Msh3 and Msh6, which function as heterodimers. The
MutSα heterodimer (Msh2-Msh6) targets single base
mispairs and single base insertion/deletions for repair,
and the MutSβ heterodimer (Msh2-Msh3), overlaps in
the repair of single base insertion/deletions, but
primarily targets larger insertion/deletions for repair [
MutS heterodimers recruit the MutL homologues (Mlh1,
Pms2, Mlh3) to mediate the next steps of repair .
Mutations in MSH2, MSH6, MLH1 and PMS2 have been
identified in Hereditary non-polyposis colorectal cancer/
Lynch Syndrome (HNPCC)/LS), a familial cancer
]. Mutations in the MSH3 gene have not been
associated with Lynch Syndrome.
The ATPase domain is highly conserved and regulates the
affinity of the MutS proteins for their DNA substrates [
Msh2 and Msh6 murine models defective in ATPase
function have been generated [
]. These mutations prevented
ATP mediated release of the DNA substrate leading to,
reduced DNA MMR, increased tumorigenesis and reduced
lifespans. The impact of an Msh3 ATPase defect has not
been determined in a murine model. The aim of this study
was to analyze the Msh3G855D mouse line, which harbors a
glycine to aspartic acid mutation in the ATPase domain of
the protein, on MMR, tumor suppression and survival.
A mutation was introduced into exon 20 of the Msh3 gene
changing codon 855 from glycine (GTT) to aspartic acid
(GAC) using site directed mutagenesis (Stategene, Quick
change kit), following sub-cloning from a bacterial artificial
chromosome. The mutation was confirmed by sequencing.
A NotI fragment containing loxP sites on either side of a
neomycin-PGK hygromycin resistance cassette was
subcloned into an EcoRI site 150 bp upstream of exon 20. The
EcoRI fragment was re-subcloned into the (+) pBluescript
vector containing Msh3 genomic DNA. The vector was
linearized and used to modify the Msh3 genomic locus via
gene targeting in G4 embryonic stem cells (ES). Positive ES
clones were identified via PCR and the correct integration
confirmed using long range PCR and southern blot
analysis. Positive ES cell clones were injected into C57BL6/6J
females (Jackson Laboratories). One transmitted the
mutant allele through its germ line. F1 males carrying the
mutant allele were mated to Zp3 Cre transgenic females
(C57BL/6J purchased from Jackson Laboratories),
resulting in deletion of the resistance cassette by loxp mediated
recombination. F1 Heterozygotes carrying the modified
allele were intercrossed producing n = 20, Msh3+/+, 30,
Msh3GD/+ and n = 22, Msh3GD/GD mice. Cohen’s effect
size value of (d = 0.7). Msh3−/− animals utilized as
negative controls were gifts from the laboratory of Dr. Winfried
]. All animals were maintained at the Albert
Einstein College of Medicine animal care facility in
accordance with Institutional Animal Care and Use Committee
Electromobility shift assay (EMSA)
The effect of the Msh3GD/GD mutation on ATPase
mediated DNA substrate release, was examined in nuclear
extracts from each genotype. Nuclear extracts were
prepared as described [
]. Nuclear extracts were prepared
from pooled mouse testes obtained from five animals of
each genotype Msh2-Msh3+/+, Msh3-Msh3GD/+,
Msh2Msh3GD/GD animals. Testes were minced, washed twice
in cold PBS and centrifuged in 15 ml conical tubes for
4 min at 4000 rpm. Pellets were re-suspended in 5
volumes of low salt buffer (10 mM HEPES, 1.5 mM MgCl2,
10 mM KCl, 0.5 mM DTT and 0.5 mM PMSF), incubated
on ice for 10 min, and centrifuged at 4 °C for 10 min at
4000 rpm. Pellets were re-suspended in 2 volumes of the
low salt buffer, homogenized in a dounce 10–20 times and
centrifuged at 4 °C for 20 min at 14,500 rpm in a Sorval.
The pellet was re-suspended in high salt buffer (20 mM
HEPES, 25% (v/v) glycerol, 420 mM NaCl, 1.5 mM MgCl2)
2 mM EDTA, 0.5 DTT, 0.5 mM PMSF,) and homogenized
3–4 times. Following homogenization, samples were
incubated for 30 min at 4 °C while stirring. The mixture was
centrifuged at 14,500 rpm in a Sorval at 4 °C for 20 min.
The supernatant was dialyzed overnight at 4C in
dialysis buffer (20 mM HEPES, 20% (v/v) glycerol, 100 mM
KCL, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF). The
supernatant was centrifuged at 14,500 rpm in a Sorval
for 20 min at 4 °C. The nuclear extracts were aliquoted
and stored at – 80 °C. To prepare binding reactions: the
sense strand oligonucleotide 5′
and antisense oligonucleotide insert
5′CCGCTGAATTG C AC C GAG C TC C AC AC AC AGAT TC C TC
GATGATCCTAAGC 3′ and homoduplex oligonucleotide 5′
CCGCTGAATTGCACCGAGCTCGATCCTCGATGATCCTAAGC were end labeled with infrared-700
(IR700) and annealed in 1× T4 Kinase buffer with 3×
molar ratio of antisense oligonucleotide containing a 4CA
insert. Thirty micrograms of nuclear extract was
pre-incubated in 1× DNA binding buffer, 1ug of poly (dI–dC) and
1 ng of unlabeled homoduplex for 5 min on ice in a total
volume of 19ul. Five nanograms of radio labeled DNA
probe was added and the mixture incubated at room
temperature in the dark for 20 min. For cold
competitor reactions, the cold competitor was added in the
preincubation reaction. ATP mediated release was induced
by adding ATP 15 min following addition of DNA. The
binding reaction mixture was electrophoresed on a 5%
polyacrylamide gel in 0.5× trisborate EDTA (TBE) buffer.
The gel was imaged using the Odyssey Infrared Imaging
System (LI-COR). Image J software was use to analyze the
binding intensities graphed in the binding curves.
Microsatellite instability analysis
Microsatellite sequences were analyzed by single cell
PCR to determine the effect of Msh3G855D mutation on
microsatellite stability. Equal amounts of tail DNA from
five mice of each genotype (Msh3+/+, Msh3GD/GD, and
Msh3−/−) were pooled separately and diluted to between
0.5 and 1.0 genome equivalents. DNA was extracted
from tumors and amplified for MSI analysis (not all
tumors amplified). PCR cycling parameters for U12235,
D17MIT91 and (TG)27 markers were performed as
previously described [
]. A 95 °C for 1 min, 57–63° C for
1 min, and 72 °C for 1 min for 30 cycles and 72 °C for
5 min once. The products were diluted by loading buffer,
heated at 95 °C for 5 min, and loaded onto 6% vertical
polyacrylamide gels. Following electrophoresis, gels were
fixed, dried, and exposed to X-ray film overnight (12 h) to
2 days. A single observer analyzed samples and a second
observer reviewed equivocal samples.
Histopathological analysis of tumors
Moribund mice were identified and sacrificed using CO2
according to IACUC protocols. Tumors were removed
and fixed in 10% neutral buffered formalin. Tumors were
then embedded in paraffin and sections were removed
for hematoxylin and eosin staining and analyzed.
The Kaplan–Meier survival curve was generated and
analyzed using Graphpad prism 3.0 software.
Microsoft Excel version 14.7.6 was used to determine the p
values between Msh2-Msh3GD/GD Msh32-Msh3+/+ and
Msh2-Msh3−/− somatic MSI using the student t test.
The Fisher exact test was used to determine the
significance of tumor MSI rates. G*Power software version
3.1 was used to analyze sample size significance.
Differences were determined to be statistical significant at p
values < 0.05.
Msh2‑Msh3 GD/GD impaired ATP mediated DNA substrate
Dissociation of the Msh2-Msh3+/+ (positive
control) and Msh2-Msh3GD/GD complexes from the DNA
substrate was observed upon addition of increasing
concentrations of cold competitor to the binding
reactions, confirming the specificity of binding (Fig. 1a).
The addition of increasing concentrations of ATP to
the binding reactions, induced release of the
Msh2Msh3+/+ heterodimer from the substrate, while the
Msh2-Msh3GD/GD heterodimer persisted at the
highest ATP concentrations (Fig. 1b). At physiological
ATP concentrations of approximately 3–5 mM [
the mutation reduced ATP induced DNA substrate
release. Preliminary titration reactions were completed
as shown in Additional file 1.
Somatic microsatellite instability in Msh2‑ Msh3 GD/GD animals
Microsatellite instability is a hallmark of MMR
]. Somatic MSI in the Msh2-Msh3GD/GD
animals was compared to Msh2-Msh3+/+ and Msh2-Msh3
−/− animals (the positive and negative controls). At the
TG27 and D7Mit91 dinucleotide markers, the highest
instability was observed in the Msh2-Msh3−/− animals,
15 and 9% respectively, with p values of 0.03 and 0.02
compared to wild type (Table 1). Conversely, no
significant instability was observed in the Msh2-Msh3GD/
GD and Msh2-Msh3+/+ animals, at the dinucleotide
markers. Indicating that the Msh2-Msh3GD/GD
complex retained MMR function at the dinucleotide repeat
sequences tested. The Msh2-Msh3−/− animals showed
no instability at the mononucleotide marker (Table 1).
While MSI in the Msh2-Msh3GD/GD animals was
significantly increased compared to Msh2-Msh3+/+ animals
at the U12235 mononucleotide marker (p value 0.03).
At the dinucleotide sequences, the Msh2-Msh3GD/GD
animals were similar to wild type and not the
Msh2Msh3−/− animals. Confirming that Msh3 MMR
function remained. Tumor MSI was tested at the U12235
(A)n and TG27 markers (no instability was observed at
the D7Mit91 marker in the tumors tested). Comparison
of MSI negative tumors from wild type animals to the
tumor numbers in the Msh2-Msh3GD/GD animals proved
significant using the Fisher exact test, p value = 0.03.
MSI in tumors was similar to the somatic MSI, with
greater instability at the mononucleotide marker in
the Msh2-Msh3GD/GD tumors and at the dinucleotide
marker in the Msh2-Msh3−/− tumors. These numbers
were not significant (Table 1).
Late stage microsatellite unstable tumors observed
with no significant loss of survival
Analysis of tumors revealed GI tumors,
lymphomas and other tumors Fig. 2a. Low tumor numbers
were consistent with a weak tumor phenotype in the
Msh2-Msh3GD/GD animals revealing no significant
difference compared to Msh2-Msh3+/+ tumorigenesis.
The Msh2-Msh3GD/GD tumors showed both
contractions and expansions (Fig. 2b). There was no
significant reduction in survival between Msh2-Msh3GD/GD
animals (21 months) compared to wild type animals
(24 months), Fig. 2c. Msh2-Msh3GD/GD animals
succumbed to tumors starting at 12th months, similar to
the tumor onset previously reported in Msh2-Msh3
−/− mice [
]. Wild type animals began to succumb
later, at 16th months (Fig. 2c).
ADP binding in the nucleotide pocket of the Msh3
ATPase domain, is thought to increase the affinity of the
Msh2-Msh3 heterodimer for DNA, while ATP binding
and hydrolysis initiates recruitment of the MutL proteins
and additional repair factors required to complete repair
and ultimately release the substrate [
in the current study a defective Msh3 ATPase domain did
not abrogate the MMR function of the Msh2-Msh3
complex. One study, suggested that the MSH3 subunit of the
heterodimer was most involved in targeting and
binding of the insertion substrate, while ATP binding in the
MSH2 subunit initiated the recruitment of the MutL
proteins to begin the final steps of repair and release [
this model would predict, the Msh2-Msh3GD/GD complex
bound to the DNA substrate, confirming that DNA
binding was not impaired by the mutation. While a defect in
ATP induced release of the CA4 substrate was confirmed.
The EMSA binding reactions were conducted in vitro
and the timeline of the binding stoichiometry was not
explored. Repair at dinucleotide sequences confirmed
Msh3 MMR function, notwithstanding the inability to
hydrolyze ATP in the Msh3GD/GD subunit. A functional
Msh2 ATPase domain appeared sufficient to promote
delayed release from the DNA substrate, facilitating the
final steps of repair in vivo.
MMR function was not significantly impaired in the
Msh3G855D mouse line. The mutation led to increased
instability at the genomic mononucleotide repeat
sequence, and in the tumors tested. Some Msh2-Msh3GD/
GD animals succumbed to these tumors at earlier ages
than did wild type animals, but the survival rates were
not significantly reduced. The results suggest that Msh3
is not a primary driver of tumorigenesis. The
Msh2Msh3GD/GD phenotype shows greater similarity to late
onset sporadic tumorigenesis and not the familial Lynch
syndrome with which mutations in the MSH2 and MSH6
MutS homologues are associated [
• ATP was added in the final 5 min of the binding
reactions and so the effect of lengthier binding reaction
times on DNA substrate release dynamics in the
presence of ATP was not evaluated.
• The inclusion of additional dinucleotide
markers, in addition to trinucleotide and tetranucleotide
genomic repeat markers would be more
informative in light of Msh3′s role in maintaining stability at
Additional file 1. Preliminary electromobility shift assay
(EMSA)—Preliminary EMSA including negative control (lane 1) and fixed concentrations of
cold competitor (30×) in lanes 5, 6 and 7. ATP (4 mM) with Msh2-Msh3+/+,
Msh2-Msh3GD/+ and Msh2-Msh3GD/GD nuclear extracts in lanes 8, 9 and 10.
Dissociation of DNA protein complexes was incomplete indicating that
concentration of DNA and competitors needed to be adjusted.
HNPCC: Hereditary non-polyposis colorectal cancer; LS: Lynch syndrome;
Msh3: MutS homologue 3; MMR: mismatch repair; MSI: microsatellite
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used during the current study are available from the
Consent for publication
The Institute of Animal Care and Use Committee (IACUC) of The Albert
Einstein College of Medicine approved all animal experiment procedures. All the
mice used were maintained in a dedicated animal care facility at the IACUC of
the Albert Einstein College of Medicine according to IACUC rodent guidelines.
All animal work was completed at the Albert Einstein College of Medicine.
This study was supported by The National Institute of Health grants CA76329
and CA93484 (WE).
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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