(Received for publication, January 17, 1996)
From the
Defects in DNA mismatch repair result in instability of simple repetitive DNA sequences and elevated levels of spontaneous mutability. The human G/T mismatch binding protein, GTBP/p160, has been suggested to have a role in the repair of base-base and single nucleotide insertion-deletion mismatches. Here we examine the role of the yeast GTBP homolog, MSH6, in mismatch repair. We show that both MSH6 and MSH3 genes are essential for normal genomic stability. Interestingly, although mutations in either MSH3 or MSH6 do not cause the extreme microsatellite instability and spontaneous mutability observed in the msh2 mutant, yeast cells harboring null mutations in both the MSH3 and MSH6 genes exhibit microsatellite instability and mutability similar to that in the msh2 mutant. Results from epistasis analyses indicate that MSH2 functions in mismatch repair in conjunction with MSH3 or MSH6 and that MSH3 and MSH6 constitute alternate pathways of MSH2-dependent mismatch repair.
Mutations in the four human mismatch repair genes hMSH2,
hMLH1, hPMS1, and hPMS2 are associated with hereditary
nonpolyposis colorectal cancer (HNPCC) ()as well as other
cancers(1, 2, 3, 4, 5, 6, 7, 8) .
Cell lines derived from these tumors are defective in DNA mismatch
repair and exhibit increased levels of spontaneous mutations and
frequent alterations of microsatellite repeat
sequences(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
An MSH2 homolog, GTBP/P160, has recently been identified in
humans. GTBP/P160 exists as a heterodimer with the hMSH2 protein, and
this complex binds to a G/T mismatch or to heteroduplexes containing a
(dT)
or (dT)
insertion on one DNA
strand(12, 13) . Cell lines that are defective in GTBP
exhibit elevated spontaneous mutability and are defective in the repair
of base-base and single nucleotide insertion-deletion mismatches (12, 14, 15) . However, the contribution of
mutations in GTBP to hypermutability and cancer predisposition
is not entirely clear at present, because two of the colorectal cancer
cell lines, DLD-1 and HCT-15 that are mutated in GTBP, also
harbor mutations in the 3`-5`-exonuclease
``proofreading'' domain of DNA polymerase
(16) . Thus, the observed phenotypes in these cell lines could
arise from the mutations in GTBP or those in pol
, or they could be due to the combined effects of the
GTBP and pol
mutations.
In Saccharomyces cerevisiae, mutations in the mismatch repair genes MSH2, PMS1, or MLH1 result in marked increases in spontaneous mutation rates and microsatellite instability (17, 18, 19, 20) . Purified MSH2 protein binds DNA containing mismatched base pairs and insertions(21, 22) , and PMS1 and MLH1 form a complex, which then binds the MSH2-DNA complex containing a mismatch(23) . Mutations in MSH3, another MSH2 homolog, cause a moderate increase in microsatellite instability (24) and have little effect on spontaneous forward mutation rates(25) . The yeast homolog of human GTBP has been identified in the yeast genome sequencing project; we refer to it as MSH6.
The manner by which MSH2, MSH3, and MSH6 proteins effect mismatch repair has not yet been determined. Studies with the human MSH2 and GTBP proteins could be interpreted to mean that while MSH2 can recognize loops larger than one nucleotide, MSH2-GTBP heterodimer is more efficient in the recognition of single base mispairs and one-base loops(12, 13, 14) . By contrast, recent studies with the yeast MSH3 gene have been interpreted to suggest that MSH2 by itself could recognize single base mispairs(24) . Overall, previously published results are in accord with the idea that MSH2 can function in mismatch repair independently of the other MSH proteins. To clarify the roles of the MSH2, MSH3, and MSH6 genes in mismatch repair and to identify the possible functional interaction among them, we have examined the effects of null mutations in these genes on microsatellite instability and spontaneous mutability and have carried out epistasis analyses. Our studies indicate that for its function in mismatch repair, MSH2 requires the MSH3 or the MSH6 gene and that MSH3 and MSH6 constitute alternate MSH2-dependent mismatch repair pathways.
Figure 1:
MSH6 is the yeast homolog of human
GTBP/p160. A, homology between the GTBP and MSH6 proteins is
shown schematically. Shaded areas represent identical or
highly conserved regions of the proteins. Spaces indicate gaps
for optimal alignment. The position of the Walker type A nucleotide
binding sequence (GKS) is indicated. aa, amino acids. B, alignment of C-terminal regions of the E. coli MutS, S. cerevisiae MSH2, MSH3, and MSH6, and human GTBP
proteins. This highly conserved region contains the Walker type A
nucleotide binding motif (GKS), indicated by asterisks.
Identical residues are boxed, and conserved residues are shaded. Amino acid positions are in parentheses. For optimal
alignment a deletion of amino acids NGKAYCV was introduced in the GTBP
sequence at the position indicated by
.
The msh6 mutation resulted in a 7-fold increase in tract
instability in pSH91 and a 6-fold increase in pSH31 (Table 1).
The msh3
mutation conferred a 32-fold elevation in tract
instability in pSH91 and a 103-fold increase in pSH31, an effect
similar to that reported recently(24) . By contrast, the msh2
mutation increases tract instability 233-fold in
pSH91 and 897-fold in pSH31 (Table 1)(17, 20) .
To determine the functional relationship between MSH3 and MSH6, we examined tract instability in strains carrying null
mutations in both these genes. Interestingly, in both plasmids pSH91
and pSH31, the rate of tract instability in the msh3
msh6
double mutant was nearly identical to the rate observed in the msh2
strain (Table 1). The synergistic increase in
the rate of tract destabilization in the msh3
msh6
double mutant over that in the msh3
or the msh6
single mutant indicates that MSH3 and MSH6 provide for alternate pathways for maintaining tract
stability. To determine the manner of interaction of MSH3 and MSH6 with MSH2, we examined tract instability in the msh2
msh3
, msh2
msh6
, and msh2
msh3
msh6
mutant strains. For both plasmid systems, we
found the rate of tract instability in these mutants to be the same as
that in the msh2
single mutant (Table 1). These
data indicate that the effect of the msh2
mutation is
epistatic to that of the msh3
and msh6
single mutations, as well as to that of the msh3
msh6
double mutation.
Our results indicate that MSH3 and MSH6 are
both required for maintaining wild type levels of tract stability and
spontaneous mutability in yeast cells; MSH3, however, has a
more prominent role in maintaining tract stability, and MSH6 plays a more active role in forward spontaneous mutability,
indicative of single bp alterations. Mutations in MSH3 and MSH6 exhibit epistasis to msh2, and the simultaneous
absence of MSH3 and MSH6 results in the same high
levels of tract instability and spontaneous mutability as in the msh2 strain. Our observations are consistent with the
following suggestions: (i) for its action in mismatch repair, MSH2 functions either with MSH3 or MSH6, and MSH2 is non-functional when both MSH3 and MSH6 are
absent, (ii) in wild type yeast cells, MSH2 and MSH3 function together in the preferential repair of 2-4-bp
insertions and deletions, whereas MSH2 and MSH6 together have a preference for removing single base mismatches,
and (iii) the absence of either MSH3 or MSH6 can be
compensated to varying extents by the other gene, depending upon the
type of mismatch, accounting for the synergistic effects observed in
the msh3
msh6
double mutant. Based on our results,
we propose the following model. The MSH2 protein combines physically
with either the MSH3 or the MSH6 protein, and the MSH2-MSH3 and
MSH2-MSH6 complexes have different substrate specificities (Fig. 2). The MSH2-MSH3 complex is more efficient at removing
2-4-bp insertions and deletions, while the MSH2-MSH6 complex is
more efficient at removing single bp mismatches. Thus, even though
purified MSH2 protein from both humans and yeast has been shown to bind
DNA containing a G/T mismatch or insertion-deletion loop-type
mismatches of up to 14 nucleotides(21, 22) , our
results predict that complex formation with MSH3 or MSH6 is obligatory
for the action of MSH2 in mismatch repair.
Figure 2: Role of MSH2, MSH3, and MSH6 proteins in mismatch repair. The MSH2 protein is proposed to form a complex with either the MSH6 or the MSH3 protein. The recognition of single nucleotide mismatches is effected by the MSH2-MSH6 complex as indicated by the thick arrow under MSH6. Small 2-4-bp insertions or deletions are ordinarily acted upon by the MSH2-MSH3 complex as indicated by the thick arrow under MSH3. However, to a limited extent, the MSH2-MSH6 complex can recognize 2-4-bp insertions and deletions, and the MSH2-MSH3 complex can recognize single nucleotide mismatches, as indicated by the dashed lines. Only in the absence of both the MSH3 and MSH6 proteins does complete loss of MSH2-dependent mismatch repair occur.
Mutations in human MSH2, MLH1, PMS1, and PMS2 account for the majority of cancers in HNPCC kindreds. However, a large proportion of sporadic colon cancers and other types of cancer do not have mutations in these genes, and no germ line GTBP mutations have been identified in HNPCC kindreds that harbored no mutations in these four mismatch repair genes(14) . Our results with the yeast MSH3 and MSH6 genes would suggest that mutations in the human MSH3 or the GTBP gene are unlikely to cause as severe a defect in mismatch repair as do mutations in MSH2. Mutational inactivation of both hMSH3 and GTBP, however, should result in increased microsatellite instability, hypermutability, and cancer predisposition characteristic of mutations in the hMSH2 gene.