From the Genetics and Molecular Biology Program, Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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Germline mutations in two human mismatch repair
(MMR) genes, hMSH2 and hMLH1, appear to account for approximately 70%
of the common cancer susceptibility syndrome hereditary nonpolyposis colorectal cancer (HNPCC). Although the hMLH1 protein has been found to
copurify with another MMR protein hPMS2 as a heterodimer, their
function in MMR is unknown. In this study, we have identified the
physical interaction regions of both hMLH1 with hPMS2. We then examined
the effects of hMLH1 missense alterations found in HNPCC kindreds for
their interaction with hPMS2. Four of these missense alterations
(L574P, K616 Mismatch repair is characterized by the recognition and repair of
mispaired nucleotides that may arise from misincorporation during DNA
replication, recombination between DNA lacking perfect homology, and
physical damage to DNA such as deamination of a methylated cytosine
(1). The best studied MMR1
system is the MutHLS or Dam-instructed pathway in Escherichia coli where mismatch repair is initiated at sites of transient undermethylation of DNA adenine
methylation (Dam) GATC sequences (for review, see Ref. 2).
Dam-instructed mismatch repair has been shown to require the
mutH, mutL, mutS, and
uvrD(mutU) genes (3, 4). The mutL gene was first
identified in the enteric bacteria Salmonella typhimurium
LT7 as a mutator (mutator LT7 or MutL) (5, 6) and later assigned as a
member of the post-replication mismatch repair machinery (3, 4). The
MutL protein was purified based on its ability to complement E. coli mutL extracts for mismatch repair (7). Although a specific
biochemical function has not yet been identified for MutL, it does
appear to interact with the mismatch recognition protein MutS (7),
leading to subsequent activation of the endonucleolytic activity of
MutH (8), which initiates excision repair by introducing a strand
scission on the unmodified strand of hemimethylated GATC sequences (9). This mechanism of methyl-directed mismatch repair appears to be unique
to Gram-negative bacteria, because homologues of MutH have not been
found in either Gram-positive bacteria or eukaryotes.
The gene coding for MutL has been highly conserved throughout evolution
(for review, see Refs. 10 and 11). Homologous genes have been found in
a number of organisms including the yeast Saccharomyces
cerevisiae where there are at least four
MutL homologues (MLH): MLH1, MLH2,
MLH3, and PMS1 (Post Meiotic
Segregation) (Ref. 11; Stanford yeast sequence data base).
Similar homologues of MutL have also been found in humans (12, 13).
Unfortunately the nomenclature has become complicated. Although human
hMLH1 is most closely related to yeast MLH1, the human hPMS1 appears most closely related to yeast MLH2 and/or MLH3, and human hPMS2 is most
closely related to yeast PMS1. Both genetic and biochemical evidence
have suggested that the S. cerevisiae MLH1 and PMS1 form a
heterodimer, and both genes have been shown to be essential for MMR
(14, 15). Copurification of the human hMLH1-hPMS2, based on its ability
to complement MMR in extracts derived from a cell line that was
genetically deficient for hMLH1, has confirmed the evolutionary
conservation of this heterodimer (16). The function and interaction of
hPMS1 as well as the yeast homologues MLH2 and MLH3 are unknown.
Defects in the human MMR pathway have been strongly implicated in the
etiology of hereditary nonpolyposis colorectal cancer (HNPCC) (12, 13,
17-19). Germline mutations in hMSH2 (human MutS homologue) and hMLH1 account for
approximately 70% of the HNPCC kindreds (20). Mutations in the
other MMR genes hPMS1, hPMS2, and hMSH6 appear rare; however, they have
been reported in a few atypical families (13, 18, 19).
Biochemical characterization and structure/function analysis of the MMR
proteins has contributed to our understanding of their contribution(s)
to hereditary and sporadic carcinogenesis. In S. cerevisiae,
an interaction region between MLH1 and PMS1 was localized to the
carboxyl termini of both proteins using a two-hybrid in vivo
assay system (21). In this study, we have localized the biochemical
interaction region of hMLH1 and hPMS2 to similar, but not identical,
carboxyl-terminal regions of both proteins. We found that the
interaction region of hMLH1 lies between amino acids 506 and 675, whereas the interaction region of hPMS2 lies between amino acids 675 and 850. To evaluate the biochemical interactions of hMLH1 mutations
found in HNPCC in vitro, we constructed several derivatives
of the hMLH1 protein that contained mutations previously reported to
cosegregate with cancer susceptibility. We found that the L574P,
K616 Reagents and Enzymes--
Restriction endonucleases and T4 DNA
ligase were purchased from New England Biolabs (Beverly, MA). PCR
reaction were performed using the High Fidelity PCR kit from Boehringer
Mannheim. Oligonucleotides were synthesized on an Applied Biosystems
(Foster City, CA) 3948 nucleic acid synthesis and purification system.
DNA plasmid constructs were purified using Qiagen (Hilden, Germany) DNA
purification kits. In vitro transcription and translation
(IVTT) reactions were performed using the Promega (Madison, WI)
TNT-coupled rabbit reticulocyte lysate system. Radiolabeled
[35S]methionine used to label proteins was obtained from
NEN Life Science Products. Glutathione-linked agarose beads were
purchased from Sigma. Tris-HCl, NaCl, Tween 20, and dithiothreitol were purchased form Amresco (Solon, OH). All other reagents used were of the
highest quality commercially available.
Subcloning of hMLH1 and hPMS2--
The cloning of hMLH1 and
hPMS2 has been previously described (12). Both hMLH1 and hPMS2 were
subcloned into pET-29a using NdeI and BamHI.
Glutathione S-transferase (GST) fusion proteins were made
using the pGEX system (Amersham Pharmacia Biotech). For ease of
subcloning, pGEX-4T-2 was modified as follows. 1) The vector DNA was
digested with EcoRI and BamHI and gel purified, and 2) the following linker was introduced by ligation (top strand, 5'-GAT CCG AGA ACC TGT ACT TCC AGG GAC ATA TGG CCA TGG GTA CCG-3'; bottom strand, 5'-AAT TCG GTA CCC ATG GCC ATA TGT CCC TGG AAG TAC AGG
TTC TCG-3'); this vector is referred to as pGEX-SG1 and allows for
subcloning using NdeI and NcoI restriction
endonuclease sites in which the ATG initiation codon is in-frame with
the GST moiety. This vector also contains a TEV protease site just
upstream of the NdeI and NcoI sites. An
NdeI restriction site (CATATG) was introduced into both
hMLH1 and hPMS2 at the initiator ATG by a method that was similar to
the introduction of an NcoI site in hMSH2 (23, 24).
pGEX-hMLH1 and pGEX-hPMS2 were constructed by digesting the pGEX-SG1
and the pET29a constructs of hMLH1 and hPMS2 with NdeI and
NotI and ligating the genes into the vector.
Truncation Mutagenesis of hMLH1 and hPMS2--
The hMLH1
deletion mutants were constructed using PCR truncation mutagenesis.
Forward primers were designed by using a codon that had a guanine in
the first position and adding the next 17 nucleotides to the following
sequence; 5'-GCG GAT CCC ATG G-3'. The reverse primers were designed by
adding the first 18 nucleotides of the complementary strand to the
following sequence; 5'-GGC ATA CTC GAG CTA-3'. Using a forward and
reverse primer, a PCR reaction was performed using pET-hMLH1 as
template. The PCR product and pET-24d were digested with
NcoI and XhoI, gel-purified, and ligated
together. The hPMS2 deletion mutants were constructed in the same
manner. The forward primers were designed by using a codon with a
guanine in the first position and adding the next 18 nucleotides to the
following sequence: 5'-GGC TAC GGT CTC CAT ATG-3'. Reverse primers were
designed by adding the first 18 nucleotides of the complementary strand
to the following sequence: 5'-CGC GGA GGA TCC CTA-3'. The PCR product
and pET-29a were digested with NdeI and BamHI,
gel purified, and ligated together. All of these constructs were
sequenced in their entirety (ABI377) to eliminate possible PCR mutagenesis.
Construction of HNPCC Mutations in hMLH1--
The HNPCC mutants
of hMLH1 were constructed by overlapping PCR using primers containing
the required mutation (24). The PCR products were gel-purified and
ligated into bluescript-hMLH1. These constructs were sequenced to
verify mutagenesis and then subcloned into pET-29a. The pET constructs
were then sequenced in their entirety to eliminate the possibility of
second site mutations.
GST Fusion Protein Interaction assay--
An overnight culture
of pGEX-hPMS2 or pGEX-hMLH1 was grown in LB medium with 50 µg/ml
ampicillin. 50 ml of LB with ampicillin was inoculated with 1 ml
overnight culture and grown to an absorbance at 600 nm of 0.5. Isopropyl-1-thio-b-D-galactopyranoside was added to a final
concentration of 0.1 mM and placed in a shaker at 30 °C
for 2 h. Induced cells were pelleted and resuspended in 800 µl
of phosphate-buffered saline (Boehringer Mannheim) plus protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.8 mg/ml
leupeptin, 0.8 mg/ml pepstatin, and 0.1 mM EDTA). Lysozyme
was added to a concentration of 1 mg/ml and left on ice for 30 min.
Triton X-100 and dithiothreitol were then added to final concentrations
of 0.2% and 2 mM, respectively, and the lysate was frozen
and thawed two times to completely lyse the cells. DNase I (Boehringer
Mannheim) was added to a final concentration of 20 µg/ml, and the
lysate was incubated on ice for 20 min. Cell debris was cleared by
centrifugation at 14,000 rpm in a refrigerated Eppendorf (Model 5402)
centrifuge for 30 min. and the supernatant was transferred to a new
microcentrifuge tube with rehydrated GST beads such that approximately
10-50 ng of protein were bound to each 25 µl of beads (see below for
quantification of GST fusion protein levels). The lysate/GST beads were
incubated at 4 °C on a rocking platform.
After rocking at 4 °C for 1-2 h, the lysate/GST beads were spun at
1,000 rpm in an Eppendorf microcentrifuge for 30 s, the supernatant was removed, and the beads were gently resuspended in 500 µl of binding buffer (20 mM Tris, pH 7.5, 10% glycerol, 150 mM NaCl, 5 mM EDTA, 1 mM
dithiothreitol, 0.1% Tween 20, 0.75 mg/ml bovine serum albumin, 0.5 mM phenylmethylsulfonyl fluoride, 0.8 mg/ml leupeptin, and
0.8 mg/ml pepstatin). The centrifugation/resuspension was repeated
three times to wash the beads free of most nonspecific lysate proteins.
The slurry was then added to a 14-ml sterile polypropylene tube,
diluted with binding buffer to approximately 50 µl of packed
glutathione beads/ml and incubated at 4 °C on a rocking platform for
30 min to allow bovine serum albumin to coat the beads. 500 µl
(10-50 ng of bound GST fusion protein) of these coated GST fusion
protein-associated glutathione beads was then aliquoted into 1.5-ml
Microcentrifuge tubes. GST fusion protein expression levels were
determined by binding the lysate to glutathione beads (as above),
followed by 3× wash and quantification of protein on Coomassie-stained
SDS-PAGE gels using bovine serum albumin as a standard (14).
IVTT (Promega) Reactions with [35S]methionine were
performed with pET-hPMS2 or pET-hMLH1 using purified DNA (Qiagen)
according to the manufacturer's recommendations. IVTT reactions were
prerun to determine the relative molar concentration of each construct. This was calculated using the specific activity of the
[35S]methionine, correcting for the number of methionines
in each IVTT construct and using SDS-PAGE and a Molecular Dynamics
PhosphorImager with ImageQuant software (Sunnyvale, CA) to quantitate
labeled peptide. Up to 10 µl of the IVTT protein was added to each
tube such that each sample had relative equimolar concentrations of IVTT protein. An IVTT reaction that used pET24d as the vector was added
to normalize the total amount of IVTT buffer/reaction mixture in each
tube. They were incubated for at least 1 h at 4 °C on a rocker.
The beads were washed three times with the binding buffer and then
resuspended in 50 µl of SDS loading buffer (0.25 Tris, pH 6.8, 5%
sucrose, 2% SDS, 5% 2-mercaptoethanol, and 0.005% bromphenol blue).
The samples were resolved on an SDS-PAGE and then imaged using a
Molecular Dynamics PhosphorImager.
Quantitation of Interaction--
The GST-IVTT interaction assay
system is not absolutely quantitative and is likely to depend on the
relative association constant (kassoc) of the
individual hMLH1 mutant proteins with hPMS2. Thus, subtle changes in
the relative concentration of interacting peptides may influence the
ultimate measure of interaction. To provide modest control between
experiments for such concentration-dependent processes, we
determined the approximate molar concentrations of the GST fusion
protein and the IVTT protein (see "Materials and Methods" above),
and each interaction experiment was designed to contain a nearly
identical ratio of the test peptides. Relative interaction
(Intrel) with hPMS2 by each missense mutant hMLH1 protein
was determined as the fraction of the mutant interaction ratio
(IRm) divided by the wild type interaction ratio
(IRwt). Intrel = IRm/IRwt. The mutant interaction ratio
IRm was determined by quantitating the amount of
interacting peptide (Molecular Dynamic PhosphorImager with ImageQuant
software) and dividing this number by the quantitated amount of
pre-experiment IVTT-expressed protein (Molecular Dynamic PhosphorImager
with ImageQuant software). This quantitation was determined (as above)
for each experiment which a priori contains all of the
mutant proteins to be tested as well as the wild type protein control
on a single SDS-PAGE gel. The wild type interaction ratio
(IRwt) was calculated similarly by quantifying the amount
of wild type IVTT protein precipitated in an interaction experiment and
dividing it by the IVTT expression control. In this way, each
interaction precipitation was normalized to the amount of protein
expressed by IVTT and introduced into the interaction experiment.
Results are presented as the mean and S.D. of four separate
experiments. These statistical methods would appear make the GST-IVTT
assay system semiquantitative in nature.
Interaction Domains of the hMLH1 and hPMS2 Proteins--
The hMLH1
and hPMS2 proteins were purified as a stable heterodimer based on
complementation of MMR in a human hMLH1-deficient cell extract (16). To
map the interaction region(s) between these proteins, we developed an
assay that relies on the use of a GST fusion protein expressed in
E. coli as a "bait" and in vitro transcribed
and translated (IVTT) protein as "prey." This assay proved to be
effective for all of the combinations that would be necessary for this
study; GST-hPMS2::IVTT-hMLH1 (Fig.
1A) and GST-hMLH1::IVTT-hPMS2
(Fig. 1B). The interaction for each of these IVTT
full-length peptides was specific for the GST fusion proteins because
we observed nearly undetectable nonspecific background binding as
demonstrated by incubation and centrifugal precipitation of the
IVTT-expressed protein with the glutathione beads alone, E. coli lysate + glutathione beads, and pGEX (the GST moiety alone) + glutathione beads as controls (Fig. 1, 2nd and 4th
lanes). Furthermore, densitometric comparison of the pGEX (only)
lane with the GST fusion (either hMLH1 or hPMS2)
lane demonstrated that the signal to background ratio in
this assay approaches 200-fold. These results suggested that this
bait-prey system was sufficient to map the interaction region(s) of the
hMLH1-hPMS2 heterodimer.
Using the GST-IVTT assay, we mapped the interaction domain of hMLH1
with hPMS2 (Fig. 2). In each experiment,
a comparison between pairs of signals obtained when IVTT-hMLH1 peptide
is added to the GST vector alone versus the GST-hPMS2 fusion
is required to determine relevant interaction. Amino acid residues
1-500 of hMLH1 did not appear to significantly interact with GST-hPMS2 (Fig. 2, pairs 2, 4, and 5). However,
significant interaction was identified in the carboxyl terminus of the
MLH1 protein (amino acids 253-756: Fig. 2, pairs 3 and
6). This apparent interaction region was further resolved by
truncating this carboxyl-terminal interaction fragment in 25 amino acid
increments. We found that amino acid residues 506-756 of hMLH1
interacted with GST-hPMS2, whereas hMLH1 amino acid residues 531-756
showed no detectable interaction (Fig. 2; pairs 6 and
7, respectively). Furthermore, hMLH1 amino acid residues
410-675 displayed interaction with GST-hPMS2, whereas hMLH1 amino acid
residues 410-650 did not (Fig. 2; pairs 8 and 9,
respectively). These results suggest that the most significant hPMS2
interaction region of hMLH1 is located between amino acid residues 506 and 675. It is worth noting that there is a significantly reduced
interaction with hPMS2 when one compares hMLH1 amino acid residues
506-756 with hMLH1 amino acid residues 410-675. These results suggest
that the interaction region of hMLH1 with hPMS2 may extend the carboxyl
terminus from amino acid residue 675.
We also determined the interaction domain of hPMS2 with hMLH1 (Fig.
3). As suggested above, a comparison
between pairs of signals obtained when IVTT-hPMS2 peptide was added to
the GST vector alone versus the GST-hMLH1 fusion is required
to determine relevant interaction. The first 600-amino acid residues of
hPMS2 did not display significant binding to GST-hMLH1 (Fig. 3; pairs 2, 4, and 5). In contrast, amino acid
residues 602-862 of hPMS2 appeared sufficient to bind GST-hMLH1 (Fig.
3; pair 6). Using the truncation strategy described above as
a mechanism to resolve the interaction region, we found that amino acid
residues 675-862 of hPMS2 bound to GST-hMLH1 (Fig. 3; pair
7) and that the interaction was lost with truncation of 25 amino acid residues (amino acids 700-862: Fig. 3; pair 8).
Similarly, amino acid residues 675-850 of hPMS2 interacted with
GST-hMLH1 (Fig. 3; pair 9), and the interaction was lost
with truncation of 25 amino acid residues (amino acids 675-825: Fig.
3, pair 10). These results suggest that the hMLH1 interaction region of hPMS2 is located between amino acid residues 675 and 850.
Effect of hMLH1 Mutations Found in HNPCC on Interaction with
hPMS2--
Several missense mutations of hMLH1 have been reported to
cosegregate with HNPCC and are found to be within the region of hMLH1
that we had determined to interact with hPMS2 (20). As a first step in
determining the functional consequences of these alterations, we have
examined hMLH1 containing the V506A, Q542L, L574P, E578G, L582V,
K616
Initial experiments suggested that several of the hMLH1 missense mutant
proteins might display subtle alterations in binding to hPMS2. To
substantiate this comparison, we developed a semiquantitative measurement of hMLH1 interaction with hPMS2 (see "Materials and Methods"). A representative experiment is shown in Fig.
4B. Six of the hMLH1 missense mutant proteins (L574P,
K616 Mutations in the human MutL homolog, hMLH1, appear to account for
approximately 35% of HNPCC patients (20). The hMLH1 protein was
purified as a heterodimer with hPMS2 protein by biochemical complementation of MMR in an hMLH1-deficient cell extract (16). Although the role of the hMLH1-hPMS2 heterodimer in MMR has not been
elucidated, it is believed to be an integral component of the MMR
machinery. These observations suggested that a disruption of the
hMLH1-hPMS2 heterodimer might block the process of MMR and manifest
itself as HNPCC.
To test whether disruption of hMLH1 and hPMS2 interaction could be a
cause of cancer susceptibility in HNPCC, we first mapped the
interaction region(s) of hMLH1 and hPMS2. We found that this interaction was confined to a single region located in the carboxyl terminus of both proteins. This observation is similar to that reported
for the respective homologues (MLH1 and PMS1) in S. cerevisiae. Comparison of the data presented here and similar
studies with the yeast homologues suggest that these heterodimers
interact through similar regions on each of these proteins. A summary
of the hMLH1 and hPMS2 interaction regions is detailed in Fig.
5. Interestingly, sequence comparison of
the five known MLH1 homologues does not reveal significant conserved
homology within the interaction region (15% identity; 38/250 amino
acids). However, sequence comparison of the five PMS1 (PMS2) homologues
(excluding hPMS1, which is largely nonhomologous) reveals significant
homology within the consensus interaction region (35% identity; 61/175
amino acids).
, R659P, and A681T) displayed >95% reduction in
binding to hPMS2. Two additional missense alterations (K618A and K618T)
displayed a >85% reduction in binding to hPMS2, whereas three
missense alterations (S44F, V506A, and E578G) displayed 25-65%
reduction in binding to hPMS2. Interestingly, two HNPCC missense
alterations (Q542L and L582V) contained within the consensus interaction region displayed no effect on interaction with hPMS2, suggesting that they may affect other functions of hMLH1. These data
confirm that functional deficiencies in the interaction of hMLH1 with
hPMS2 are associated with HNPCC as well as suggest that other unknown
functional alteration of the human MutL homologues may lead to
tumorigenesis in HNPCC kindreds.
INTRODUCTION
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Abstract
Introduction
References
, K618A, K618T, R659P, and A681T missense mutations of hMLH1
displayed a >80% loss of interaction with hPMS2, whereas the S44F,
V506A, and E578G missense mutations displayed a 20-80% reduction in
interaction with hPMS2. Two missense alterations (Q542L and L582V)
showed no reduction in hPMS2 interaction, and one of those (Q542L) was
also found to have no effect in the dominant mutator assay (22). These
data indicate that loss or reduced interaction between hMLH1 and hPMS2
may play a causative role in the development of HNPCC. However, other
as yet undefined functional alterations also appear to contribute to
cancer susceptibility. These data are largely consistent with an
in vivo dominant mutator assay developed in yeast that was
used to examine mutations of hMLH1 found in HNPCC (22).
MATERIALS AND METHODS
RESULTS
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Fig. 1.
GST fusion protein assay to study the
interaction domain between hMLH1 with hPMS2. Labeled
([S35]methionine) IVTT protein was precipitated using
hPMS2 (panel A)- or hMLH1 (panel B)-GST fusion
protein. IVTT control, 10% of the total IVTT mixture used
in each experiments; +glutathione beads, IVTT protein added
to glutathione beads (only); +E. coli lysate, glutathione
beads pretreated with an E. coli lysate similar to lysates
from which the GST moiety and the GST-hMLH1 or GST-hPMS2 proteins were
isolated; +pGEX lysate, glutathione beads pretreated with an
E. coli lysate that had been induced for expression of GST
moiety (alone) from the vector pGEX-SG1;
+pGEX-h(MLH1 or PMS2), glutathione
beads pretreated with E. coli lysates induced for expression
of either a GST-hMLH1 or GST-hPMS2 fusion protein. Samples were
resolved on an 8% SDS-PAGE and imaged using a Molecular Dynamics
PhosphorImager. No other bands were visible on the gels other than
those shown.
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Fig. 2.
Interaction region of hMLH1 with hPMS2.
S35-Labeled full-length and truncation mutants of
IVTT-hMLH1 were added to glutathione beads that had been pretreated
with either GST (alone) or GST-hPMS2. Panel A, phosphorimage
of samples resolved on a 10% SDS-PAGE. Panel B,
illustration of the constructs that were used in this experiment and
corresponding domain locations. The numbers correspond to
the pairs shown in panel A. The consensus hPMS2 interaction
domain is shaded black in the full-length hMLH1 (panel
B, 1).
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Fig. 3.
Interaction region of hPMS2 with hMLH1.
S35-Labeled full-length and truncation mutants of
IVTT-hPMS2 were added to glutathione beads that had been pretreated
with either GST (alone) or GST-hMLH1. Panel A, phosphorimage
of samples resolved on a 11% SDS-PAGE. Panel B,
illustration of the constructs that were used in this experiment and
corresponding domain locations. The numbers correspond to
the pairs shown in panel A. The consensus hMLH1 interaction
domains are shaded gray in the full-length hPMS2
(panel B, 1)
, K618A, K618T, R659P, and A681T missense mutations for their
interaction with hPMS2. We also tested the S44F mutation, because
alteration of the homologous residue in S. cerevisiae has
been reported to effect the interaction of yeast Mlh1p with Pms1p (21).
In each of four separate experiments, we determined the relative IVTT
protein expression (Fig. 4A)
and exposed equivalent molar quantities of IVTT protein to identical quantities of GST-hPMS1 for each interaction pair under study. To rule
out selective degradation of mutant proteins, we assessed the stability
of the missense mutant proteins in the glutathione agarose-GST-lysate
by incubating the IVTT proteins for 5 h under interaction
conditions (a normal interaction incubation is 1 h) and then
resolving an aliquot by SDS-PAGE. We found no detectable degradation of
the mutant peptides during this 5-fold-longer incubation period (data
not shown). Interestingly, this lack of degradation could be
demonstrated for the full-length protein as well as the numerous
internal-start peptides that constitute the bulk of the background
lower molecular weight material. Thus, it is unlikely that any
reduction in interaction with hPMS2 is because of either altered
expression levels or decreased stability of the mutated proteins.
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Fig. 4.
Effect of HNPCC mutations on hMLH1
interaction with hPMS2. Analysis of the interaction of hMLH1
missense mutations reported to cosegregate in HNPCC kindreds with
hPMS2. Panel A, the total S35-labeled IVTT
expressions of each of the mutants resolved on 8% SDS-PAGE and imaged
with a PhosphorImager (Molecular Dynamics). Panel B,
peptides were resolved on 8% SDS-PAGE and examined by PhosphorImager
(Molecular Dynamics). Panel C, the relative interaction of
each of the HNPCC mutants was determined by using ImageQuant software
and correcting for total IVTT expression of each mutant (see
"Materials and Methods"). Results from four separate experiments
are shown with S.D. error bars.
, K618A, K618T, R659P, and A681T) showed >85% reduction in
their interaction with hPMS2. The S44F, E578G, and V506A MLH1 missense
mutant proteins displayed 25-65% of that observed with wild type
interaction with hPMS2 (26.5 ± 7.1%, 54.9 ± 9.2%, and
65.7 ± 10.9%, respectively). Two hMLH1 missense mutant proteins,
Q542L and L582V, appeared to have little effect on interaction with
hPMS2 (102.5 ± 2.7% and 101.5 ± 30% of wild type
interaction, respectively). It is interesting to note that two
different hMLH1 missense mutations at residue 618 (K618A and K618T) had
nearly identical effects on their interaction with hPMS2 (11.8 ± 2.5% and 5.3 ± 1.3% of wild type, respectively), thus reducing
the likelihood that the altered interactions observed were artificial.
DISCUSSION
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Fig. 5.
Model of the hMLH1 and hPMS2
interaction. The interaction region of hMLH1 with hPMS2 is shown
in gray, and they are connected with lines. The
location of the HNPCC mutations in hMLH1 that were tested in these
studies and found to have a greater than 80% reduction in binding to
hPMS2 are illustrated as black diamonds. HNPCC mutations
that had a 20-80% reduction in binding to hPMS2 are shown as
gray diamonds. The HNPCC mutations, which had no effect on
binding to hPMS2, are shown as white diamonds. Amino acids
675-756 of hMLH1 are contained in a gray cross-hatch box to
identify it as a potential extension of the interaction region based on
the significant (though not total) reduction in interaction found with
the 475-675 hMLH1 truncation (see Fig. 2, lane 8).
We found a strong correlation between HNPCC mutations and a loss of
hMLH1 interaction with hPMS2. We tested 11 hMLH1 missense alterations
reported in HNPCC kindreds and located in the consensus interaction
domain of hMLH1. Nine of these missense alterations displayed reduced
interaction with hPMS2. Four of these alterations (L574P, K616D, R659P,
and A681T) displayed nearly undetectable interaction with hPMS2
(>99%), whereas two alterations (K618A and K618T) displayed a
significant loss of interaction (>85%). Because gross alteration of
protein structure can not be eliminated and is likely to be
functionally significant for several of these missense mutations, our
observations with the K618T and K618A alterations provide significant
foundation for the idea that protein structure is not the only
contributor to heterodimer protein interaction. This notion is
underlined by the observation that the K616 alteration (which is
located in the first of three lysine residues that includes Lys-618)
displays nearly undetectable interaction. Thus, it is appears possible
that one or all three of these lysine residues form a charge-based
contact between hMLH1 and hPMS2.
Although the S44F alteration of hMLH1 was found to be outside of the consensus hPMS2 interaction region, we tested this missense alteration in our system because the homologous alteration in S. cerevisiae displayed reduced interaction with PMS1 (21). We found a reproducible reduction in the ability of S44F hMLH1 to interact with hPMS2 (26.5% of wild type). We consider three possibilities. 1) The GST-IVTT assay for mapping these regions may have failed to detect a possible interaction domain at the amino terminus of hMLH1, where the fusion is located, 2) the S44 amino acid may be in close spatial proximity to the consensus hPMS2 interaction region, or 3) substitution of Ser-44 with a phenylalanine gravely disrupts the protein structure such that the downstream interaction region is largely affected.
The V506A and E578G hMLH1 missense alterations, both, appeared to display consistent reduced binding to hPMS2. Although it is still possible that these alterations do not alter interaction with hPMS2 in vivo, it is interesting to note that a functional defect has been confirmed for these alterations using a yeast dominant mutator assay that would appear to depend on similar protein-protein interactions (22).
Two hMLH1 missense alterations, Q542L and L582V, did not appear to affect interaction with hPMS2. These alterations have been reportedly identified in patients meeting the Amsterdam criteria for HNPCC (25, 26). We can not rule out the possibility that an altered interaction by these amino acid changes is obscured by the amino-terminal GST fusion of these constructs. However, more provocative possibilities are that the Q542L and L582V may be either nonfunctional polymorphisms or that these alterations affect a function of the hMLH1 protein other than interaction with hPMS2. Interestingly, the Q542L missense alteration has no effect in an independent yeast dominant mutator assay system (22). Because these alterations are reportedly from well defined HNPCC kindreds, these results appear to support the notion that there are other functional alterations of the human MutL homologues that may contribute to cancer susceptibility.
The 11 hMLH1 proteins containing missense alterations appear to establish a strong link between HNPCC and loss of interaction with hPMS2. It is interesting to note that there have been no reports of single-amino acid substitutions in hPMS2, which are located in the hMLH1 interaction domain. This observation may suggest that there is another molecular partner for hMLH1 that has a redundant function with hPMS2 (similar to the findings with hMSH3 and hMSH6). hPMS1 could be a candidate for this role.
We have previously identified the interaction domains of hMSH2 with
hMSH3 and hMSH2 with hMSH6 (27). However, we found no effect of
missense alterations found in HNPCC kindreds on the interaction with
either hMSH3 and hMSH6. This observation suggested that alteration of a
static interaction between hMSH2 with hMSH3 or hMSH6 was unlikely to
play a causative role in HNPCC. Here we confirm the correlation between
altered interaction between hMLH1 and hPMS2 and susceptibility to
HNPCC.
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ACKNOWLEDGEMENTS |
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We thank Hansjuerg Alder and the employees of the Sidney Kimmel Nucleic Acid Facility for nucleotide synthesis and sequencing, Christoph Schmutte for helping to prepare the figures for this manuscript, and Teresa Wilson, Scott Gradia, and Greg Tombline for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA56542 and CA67007.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Kimmel Cancer Center
BLSB933, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-1345; Fax: 215-923-1098; E-mail: rfishel{at}hendrix.jci.tju.edu.
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ABBREVIATIONS |
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The abbreviations used are: MMR, mismatch repair; h-, human; HNPCC, hereditary nonpolyposis colorectal cancer; IVTT, in vitro transcription and translation; DAM, DNA adenine methylation; MSH, MutS homologue; MLH, MutL homologue; PMS, post meiotic segregant; kassoc, relative association constant; Intrel, relative interaction; IRm, mutant interaction ratio; IRwt, wild type interaction ratio; GST, glutathione S-transferase; mutH, mutL, mutS, MutHLS; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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