From the Sealy Center for Molecular Science,
University of Texas Medical Branch, Galveston, Texas 77555-1061 and
the § Department of Molecular Medicine, Institute of
Biotechnology University of Texas Health Science Center, San
Antonio, Texas 78245-3207
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MSH2 and MSH6 proteins exist as a stable complex,
as do the MLH1 and PMS1 proteins. To study the mismatch binding
properties of the MSH2-MSH6 complex and to examine its functional
interaction with the MLH1-PMS1 complex, these protein complexes were
purified to near homogeneity from overproducing yeast strains. As has
been reported previously, the purified MSH2-MSH6 complex binds DNA substrates containing a G/T mismatch and insertion/deletion mismatches, but the binding affinity for the latter decreases as the size of the
extrahelical loop increases. Addition of ATP or the nonhydrolyzable ATPS reduces binding of the MSH2-MSH6 complex to the DNA substrates markedly. Here, we show that MSH2-MSH6 forms a ternary complex with
MLH1-PMS1 on a mismatch containing DNA substrate. The formation of this
ternary complex requires ATP, which can be substituted by ATP
S,
suggesting that ATP binding alone is sufficient for ternary complex
formation. Thus, it appears that ATP binding by the MSH2-MSH6 complex
induces a conformation that is conducive for the interaction with
MLH1-PMS1 complex, leading to the formation of the ternary complex.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mismatch repair in yeast and other eukaryotes requires a number of evolutionarily conserved protein factors including three Escherichia coli MutS homologs, MSH2, MSH3, and MSH6, and two E. coli MutL homologs, MLH1 and PMS1 (1-3). Genetic studies in the yeast Saccharomyces cerevisiae have indicated that mutations in the MSH2, MLH1, and PMS1 genes result in severe defects in mismatch repair, whereas mutations in the MSH3 or MSH6 genes engender a much smaller mismatch repair defect. From these studies, it has been inferred that MSH2 protein functions in mismatch repair in conjunction with MSH3 or MSH6, and in the absence of the latter two proteins, MSH2 is inactive (4-6). Biochemical studies with yeast and human proteins have clarified the role of MSH2, MSH3, and MSH6 in the mismatch recognition reaction. MSH2 dimerizes with either MSH3 or MSH6, and the resulting MSH2-MSH3 and MSH2-MSH6 protein complexes have somewhat different mismatch binding specificities. MSH2-MSH6 binds the G/T mismatch and small extrahelical DNA loops preferentially, whereas the MSH2-MSH3 complex shows higher binding affinity for larger extrahelical DNA loops (7-11). MSH2 protein alone has little affinity for DNA mismatches (8).
The aforementioned biochemical studies have yielded important information concerning the role of MSH2, MSH3, and MSH6 proteins in DNA mismatch recognition. Despite their indispensability in mismatch repair, relatively little is known about the roles of the MLH1 and PMS1 proteins in the repair process. Recently, we overproduced and purified the MLH1-PMS1 complex from a yeast strain genetically tailored to co-overexpress the two proteins (12). Our biochemical studies indicated that whereas MLH1-PMS1 has no affinity for the DNA mismatch, it forms a ternary complex with MSH2-MSH3 on the DNA mismatch and also enhances the ability of the latter to bind the DNA mismatch (12). The addition of ATP has no effect on the binding of MLH1-PMS1 to MSH2-MSH3 mismatch complex (12). Here we examine the interaction between MLH1-PMS1 and MSH2-MSH6 protein complexes with the DNA mismatch. Interestingly, we find that ternary complex formation among these entities requires ATP.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Polyclonal Antibodies Specific for MSH2, MSH6, MLH1, and PMS1-- The portion of the MSH6 gene encompassing amino acid residues 38 to 612 of the encoded protein was fused to the E. coli transcription terminator rho, and the resulting insoluble rho-MSH6 fusion polypeptide was purified by preparative SDS-polyacrylamide gel electrophoresis, dialyzed against phosphate-buffered saline (10 mM NaH2PO4, pH 7.2, 150 mM KCl), and used for raising polyclonal antiserum in rabbits. Antibodies were affinity-purified from the antiserum using a column of cyanogen bromide-Sepharose containing the cross-linked antigen. Antibodies specific for the MSH2, MLH1, and PMS1 proteins were obtained as described (8, 12). All the affinity purified antibodies were dialyzed against phosphate-buffered saline and concentrated to 2 mg/ml using Centricon-30 microconcentrator to be used in the supershifting experiment.
Yeast Plasmids--
The MSH2 protein coding sequence
from the ATG translation start codon to 259 nucleotides downstream of
the TAA stop codon was placed under the control of the
galactose-inducible GAL-PGK promoter to yield plasmid
pMSH2.1 (2µ, GAL-PGK-MSH2, URA3). The MSH6 gene
from the ATG translation start to 1445 bases downstream of the
terminator codon was also placed under the control of the GAL-PGK promoter to yield plasmid pMMR10 (2µ,
GAL-PGK-MSH6, leu2d). These plasmids were introduced into the
protease-deficient yeast strain BJ5464 (MAT, ura3-52, trp1,
leu2-
1, his3-
200, pep4::HIS3, prb1
1.6R, can1,
GAL). The plasmids pMMR75 (2µ, ADC-MLH1,TRP1) and
pMMR97 (2µ, GAL-PGK-PMS1, leu2d), used for the
overexpression of the MLH1 and PMS1 proteins, have been described
previously (12).
Purification of the MSH2-MSH6 and MLH1-PMS1 Protein
Complexes--
Yeast strain BJ5464 harboring the plasmids pMSH2.1 and
pMMR10 was grown overnight in synthetic medium lacking both leucine and
uracil and containing 3% glycerol, 2% lactic acid, and 2% sucrose.
The overnight culture was diluted to 0.2 A600 in
the same medium containing no sucrose and then grown for 16 h. The expression of MSH2 and MSH6 proteins was induced by the addition of 2%
galactose to the medium for 7 h. The MSH2-MSH6 complex was purified using a procedure modified from that of Alani (11). Briefly,
3.4 g of cells were broken in 3 volumes of buffer A (25 mM Tris, pH 7.6, 1 mM EDTA, 10 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride)
containing 0.2 M NaCl using a French press. The extract was
clarified by ultracentrifugation (90 min at 100,000 × g), and the supernatant was loaded onto a PBE- 94 column
(1 × 4-cm). The bound proteins were eluted by a 50 ml 0.2-1
M NaCl gradient in buffer A. The fractions containing the
MSH2-MSH6 complex were diluted 3 times before being loaded onto a
single-stranded DNA cellulose column (1 × 1.5-cm; United States
Biochemical). The proteins were eluted by a 10-ml 0.2-1 M
NaCl gradient in buffer A; the fractions containing the MSH2-MSH6
complex were diluted to bring the conductivity of the pool to an
equivalent to 100 mM NaCl before being loaded onto Mono S
(HR5/5). The bound proteins were eluted with a 20-ml 0.1-1
M NaCl gradient in buffer A. Purified MSH2-MSH6 complex was
concentrated in an Amicon-30, divided in small portions, and frozen at
70 °C .
DNA Mobility Shift Assay--
All of the DNA substrates shared
the same bottom strand, which was 5'-end-labeled with
[-32P]ATP and T4 polynucleotide kinase (Fig.
2A). Because of the shared bottom strand, all of these DNA
substrates had the same specific radioactivity (1.2 × 106 cpm/µg). The substrates (5 ng, 6000 cpm) were
incubated for 30 min on ice with the indicated amounts of the MSH2-MSH6
and MLH1-PMS1 complexes, and 80 ng of HaeIII-digested
X174 double-stranded DNA added as nonspecific competitor in 10 µl
of reaction buffer (30 mM Tris-HCl, pH 8.0, 1 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 50 µg/ml of bovine serum albumin, and
10% glycerol). After the addition of 3 µl of gel loading buffer (10 mM Tris-HCl, pH 7.5, 3 mM EDTA, 50% glycerol),
the reaction mixtures were run in a 4% nondenaturing polyacrylamide
gel in 40 mM Tris acetate, pH 7.5, containing 2 mM EDTA. After drying, the gels were exposed to x-ray films
and were also subjected to phosphoimage analyses in a Molecular
Dynamics PhosphorImager. For the antibody supershifting experiment in
Fig. 4, 2 µg of affinity purified antibodies in phosphate-buffered
saline were added at the end of the incubation, and the reaction
mixtures were kept at 0 °C for 10 min before being subjected to gel
electrophoresis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MSH6 protein exists as a stable complex with the MSH2 protein in both yeast and human (7, 10, 11). To purify the MSH2-MSH6 complex for biochemical studies, we fused the MSH2 and MSH6 genes to the GAL-PGK promoter to yield plasmids pMSH2.1 (GAL-PGK-MSH2, URA3) and pMMR10 (GAL-PGK-MSH6, leu-2d), respectively. These plasmids were introduced into the protease-deficient yeast strain BJ5464, and the overexpression of MSH2 and MSH6 proteins was verified by immunoblot analyses (Fig. 1A) using affinity purified antibodies raised against portions of the two MSH proteins expressed in and purified from E. coli (see "Materials and Methods"). For the purification of the MSH2-MSH6 complex, extract from BJ5464 harboring pMSH2.1 and pMMR10 was subjected to chromatographic fractionation in columns of PBE-94, single-stranded DNA cellulose, and Mono S, as described under "Materials and Methods." When the MSH2-MSH6 complex from the last step of purification in Mono S was analyzed on a 7.5% polyacrylamide gel and stained with Coomassie Blue, only the MSH2 and MSH6 protein bands were seen (Fig. 1B), indicating that the protein preparation was nearly homogeneous.
|
For examining the mismatch binding properties of the purified MSH2-MSH6 complex, we hybridized oligonucleotides to yield duplex substrates (Fig. 2A) that contained no DNA mismatch (G/C), a G/T mismatch (G/T), and extrahelical loops of 1 base (+1), 2 bases (+2), and 4 bases (+4). These substrates were incubated with the MSH2-MSH6 protein complex, and the reaction mixtures were analyzed on 4% nondenaturing polyacrylamide gels run at 4 °C. Nucleoprotein complexes were visualized by autoradiography of the dried gels, and quantification of binding was done by phosphoimage analysis of the gels. As shown in Fig. 2, B and C, preferential binding of the MSH2-MSH6 complex to the mismatch containing DNA substrates was seen. However, different levels of nucleoprotein complex formation were observed with the DNA substrates, and the results revealed that the MSH2-MSH6 complex has the highest affinity for the +1 substrate, followed by the G/T, +2, and +4 substrates and that only a trace amount of the homoduplex (G/C) was bound (Fig. 2, B and C). Thus, by contrast to the MSH2-MSH3 complex, which has a higher affinity for DNA substrates containing larger extrahelical loops (8, 9, 12), the MSH2-MSH6 complex binds less well to DNA containing larger extrahelical loops. With all the mismatched DNA substrates, MSH2-MSH6 forms two nucleoprotein complexes (Fig. 2B). The reason for these two shifted forms is not known.
|
One rather intriguing aspect concerning the mismatch recognition
ability of the MSH2-MSH6 complex is its marked sensitivity to ATP (7,
10, 11), which is not seen with the MSH2-MSH3 complex (8, 12). For
instance, the inclusion of 1 mM ATP to the binding
reactions diminishes binding to the G/T and +1 substrates markedly
(Fig. 2, D and E). In other experiments, an inhibition of DNA mismatch binding by the MSH2-MSH6 complex was also
seen when ATPS instead of ATP was added to the reaction mixture (see
Fig. 3C). Thus, it appears
that the ATP bound form of the MSH2-MSH6 complex has a much lower
affinity for DNA mismatch.
|
In addition to MSH2, MSH3, and MSH6 proteins, mismatch repair is also dependent on the MLH1 and PMS1 gene products. The MLH1 and PMS1 proteins also exist as a stable stoichiometric complex in yeast cells, and the complex of these proteins has been purified from a yeast strain genetically tailored to co-overexpress the two proteins (12), and the purified MLH1-PMS1 complex used in this study was nearly homogeneous (Fig. 1B). As reported in our recent work (12) and reiterated here in Fig. 3A, no binding of MLH1-PMS1 protein complex to the homoduplex or to any of mismatch-containing DNA substrates (G/T, +1, +2, and +4) was evident. At much higher concentrations of MLH1-PMS1 than those used in the experiments reported here, a low level of nonspecific binding to all of the DNA substrates occurs, but under no circumstances have we detected preferential binding to the DNA mismatch. Taken together, these results indicate that the MLH1-PMS1 complex has no mismatch recognition ability.
We showed recently that the MLH1-PMS1 complex enhances the mismatch binding ability of the MSH2-MSH3 complex and forms a ternary complex with the latter on the DNA mismatch (12). Because of this result, it was of considerable interest to examine the effect of the purified MLH1-PMS1 complex on the mismatch binding ability of the MSH2-MSH6 complex, and whether a ternary complex between these two protein complexes is formed on the DNA mismatch. As shown in Fig. 3A, the addition of amounts of MLH1-PMS1 complex stoichiometric to that of MSH2-MSH6 complex had no significant effect on the mismatch binding ability of the latter protein complex. In Fig. 3A, the +1 substrate was used for testing the effect of the MLH1-PMS1 complex. We have also used the G/T substrate and found no significant effect of the MLH1-PMS1 complex on the binding of this DNA mismatch substrate by MSH2-MSH6 or any evidence of the formation of a ternary complex between the DNA mismatch and MLH1-PMS1 and MSH2-MSH6 protein complexes.
The results in Fig. 3A produced no evidence for a functional or physical interaction between the MSH2-MSH6 complex and the MLH1-PMS1 complex, which was a somewhat surprising result, given that genetic and biochemical studies have indicated that MSH2-MSH6-dependent mismatch repair also requires the MLH1-PMS1 complex, and that the latter has been found to interact with the other mismatch recognition protein complex, MSH2-MSH3, in the presence of a DNA mismatch forming a ternary complex (12). Since ATP binding apparently induces a substantial conformational change in MSH2-MSH6 as indicated by a highly reproducible reduction in the binding of DNA mismatches (Fig. 2, D and E), we reasoned that perhaps this ATP-induced conformational change in the MSH2-MSH6 complex is also a prerequisite for interaction with the MLH1-PMS1 complex. To test this idea directly, we repeated the mismatch binding experiment with both the MLH1-PMS1 and MSH2-MSH6 protein complexes and the G/T and +1 substrates, but in the presence of 1 mM ATP (Fig. 3B), which as shown previously (Fig. 2, D and E) effects a large reduction in the binding of the MSH2-MSH6 complex to these DNA. Interestingly, a novel nucleoprotein complex (designated by C*) which migrates above the normal MSH2-MSH6-mismatch nucleoprotein complex (designated by C) was formed with both the G/T and +1 substrates in the presence of ATP.
To determine if ATP binding alone was sufficient or if ATP hydrolysis
was also required for ternary complex formation, we examined whether
the novel nucleoprotein complex was formed in the presence of the
nonhydrolyzable ATP analogues ATPS and
AMP-PNP1 (Fig.
3C). As has been reported previously for the human MSH2-MSH6 complex (7), addition of AMP-PNP did not reduce the mismatch binding
ability of the yeast MSH2-MSH6 complex (Fig. 3C, lane 6). AMP-PNP also did not promote the formation of a ternary
complex of MSH2-MSH6 with MLH1-PMS1 on mismatched DNA (Fig.
3C, lane 12). The lack of any effect of AMP-PNP
may result from the inability of MSH2-MSH6 to bind this nucleotide, or
binding to this nucleotide may not alter the conformation of MSH2-MSH6
to a form appropriate for interaction with MLH1-PMS1. As shown in Fig.
3C, ATP
S, however, induced the formation of the novel
nucleoprotein complex, albeit at a somewhat lower efficiency than ATP,
suggesting that binding of ATP by MSH2-MSH6 is sufficient to promote
ternary complex formation.
In a recent study (13), it has been suggested that the human MSH2-MSH6 complex cycles between ADP and ATP bound forms. According to these workers, the ADP bound form of human MSH2-MSH6 binds the DNA mismatch and serves as a nucleation point for the assembly of other mismatch repair proteins. Turnover of the human MSH2-MSH6 complex from the mismatch is triggered upon the exchange of ADP to ATP by the protein complex. Our results with the yeast MSH2-MSH6 heterodimer, however, have indicated that ADP does not induce the formation of a ternary complex of MSH2-MSH6 with MLH1-PMS1 on mismatched DNA. Similar to the effect of AMP-PNP (Fig. 3C), addition of 1 mM ADP had no effect on the mismatch (G/T) binding ability of the MSH2-MSH6 complex, and it did not promote the formation of the ternary nucleoprotein complex (data not shown). Thus, our observations in yeast indicate the involvement of the ATP bound form of MSH2-MSH6 in mismatch repair.
The availability of antibodies against the various mismatch repair proteins has allowed us to directly test for the presence of the MLH1 and PMS1 proteins in the ternary complex by antibody-mediated supershifting. As shown in Fig. 4, treatment of the ternary nucleoprotein complex with either affinity purified anti-MLH1 or anti-PMS1 antibodies resulted in a retarded mobility of this complex, whereas treatment of the binary nucleoprotein complex formed between MSH2-MSH6 and the DNA mismatch with these antibodies had no effect on its gel mobility. As expected, treatment of the ternary complex with anti-MSH2 antibodies also resulted in retarded mobility of the nucleoprotein complex. In the absence of ATP, no evidence of ternary complex formation was seen, as evidenced by the lack of supershifting by the anti-PMS1 or anti-MLH1 antibodies of the DNA mismatch bound MSH2-MSH6 complex that had been incubated with the MLH1-PMS1 complex (data not shown). Taken together, our results indicate that ATP binding promotes the formation of a ternary complex of MLH1-PMS1 with mismatch bound MSH2-MSH6.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have purified the MSH2-MSH6 protein complex from yeast and carried out biochemical studies to examine the mismatch binding properties of the MSH2-MSH6 complex and explore the functional and physical interactions of this complex with the MLH1-PMS1 protein complex. As has been reported previously (7, 10, 11), the MSH2-MSH6 complex binds with higher affinity to DNA substrates that contain a single-base mismatch and small extrahelical loops than to those that contain larger loops. The mismatch binding preference of the MSH2-MSH6 complex differs from that of the MSH2-MSH3 complex, which has higher avidity for substrates with larger extrahelical loops (8, 9, 12).
The mismatch binding activity of the MSH2-MSH6 protein complex is
highly sensitive to ATP or the nonhydrolyzable analogue ATPS,
suggesting that nucleotide binding induces a marked conformational change in the MSH2-MSH6 complex. One interpretation of these results is
that ATP binding by the mismatch bound MSH2-MSH6 complex effects the
release of the protein complex from the DNA mismatch. Given that we saw
no evidence of ternary complex formation among MSH2-MSH6, MLH1-PMS1,
and the DNA mismatch in the absence of ATP, we considered the
possibility that the ATP-induced conformational change in the MSH2-MSH6
protein complex may be a prerequisite for the interaction of the former
with the MLH1-PMS1 protein complex. Indeed, the addition of ATP
promotes the formation of a ternary complex of MLH1-PMS1, MSH2-MSH6,
and the DNA mismatch. We have also shown that ATP
S can effectively
substitute for ATP, suggesting that nucleotide binding by MSH2-MSH6 is
sufficient for ternary complex formation. In its dependence upon ATP
for ternary complex formation, the MSH2-MSH6 complex resembles
the E. coli MutS protein, where the binding of the
MutSmismatch complex to MutL also requires ATP binding but not its
hydrolysis (14).
In summary, the MLH1-PMS1 complex combines with either the MSH2-MSH3 complex (12) or the MSH2-MSH6 complex (this work) and the DNA mismatch to form a ternary complex. In the case of MSH2-MSH3, ternary complex formation is not dependent on ATP (12), whereas with MSH2-MSH6, ATP binding is required for the assembly of the ternary complex (this work). We suggest that the ternary complexes consisting of MLH1-PMS1 and either MSH2-MSH3 or MSH2-MSH6 would be critical for the recruitment of other components indispensable for mismatch repair including the proliferating cell nuclear antigen (15, 16).
![]() |
FOOTNOTES |
---|
* This work was supported by Grants CA35035 and CA41261 from National Cancer Institute, GM19261 from National Institutes of Health, DE-FG03-93ER61706 from the Department of Energy, and ES07061 from the National Institute of Environmental Health Sciences.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: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., Galveston, TX 77555-1061. Tel.: 409-747-8602; Fax: 409-747-8608; E-mail: sprakash{at}scms.utmb.edu.
1
The abbreviation used is: AMP-PNP, adenosine
5'-(,
-imino) triphosphate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|