Download Chapter 2: DNA mismatch repair

Chapter 2: DNA mismatch repair
Mismatch repair in bacteria
Maintenance of genomic stability requires proper replication, recombination, and repair
processes. During DNA replication and recombination or by chemical modification, DNA
mismatches may arise. In the repair of these mismatches, so-called mismatch repair (MMR)
systems play a prominent role. The best characterised MMR system is that of Escherichia
coli, also known as the MutHLS system (Modrich, 1991; 1994). Three proteins, MutS, MutL,
and MutH play a crucial role in this system (Table 4). They participate in a methyl-directed
pathway, which corrects base-base mismatches and small insertions/deletions that result from
DNA polymerase slippage during DNA replication (Friedberg et al., 1995; for review see
Crouse 1997). In this methyl-directed pathway, the MutS protein recognises and binds to
DNA mismatches. Mismatch-bound MutS then forms a complex with a MutL protein. This
complex is activated by binding of the MutH protein. Besides MutS, MutL, and MutH, several
other proteins are known to be necessary for mismatch repair, such as MutU/UvrD helicase II,
RecJ or ExoVII as a 5’-exonuclease, Exo1 as a 3’-exonuclease, DNA polymerase III, singlestrand binding protein, and DNA ligase (Modrich, 1991; 1994). MutH serves as an
endonuclease by generating a nick at a GATC site on the unmethylated DNA strand near the
mismatch (Fig. 2). The strand spanning the nick and the mismatch is excised by a 3’-5’ or 5’3’ exonuclease and replaced by a new DNA strand synthesised by a DNA polymerase. In
bacteria with a defective MMR system mutations accumulate. Such bacteria have a so-called
mutator phenotype.
Table 4. Comparison of DNA mismatch repair genes
in bacteria, yeast and humans
Escherichia coli
MutS
Saccharomyces cerevisiae
Homo sapiens
MSH1a
MSH2
hMSH2
MSH3
hMSH3 (Rep-3, DUG1)
MSH4a
MSH5
hMSH5
MSH6
hMSH6 (GTBP, p160)
MutL
MLH1
hMLH1
MLH2b
hPMS1
PMS1b
hPMS2
MutH
?
?
Exo1
EXO1
hEXO1
a: Human homologues for S. cerevisiae MSH1 and MSH4 have not been published.
b: hPMS1 and hPMS2 are related to S. cerevisiae MLH2 and PMS1 respectively.
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Mismatch repair in yeast
The MutHLS system of E. coli seems to have been conserved throughout evolution (Kolodner,
1996; Modrich & Lahue, 1996). Six MutS homologues, products of the genes MSH1-6 and
three MutL homologues, products of the genes MLH1, MLH2 and PMS1 have been identified
in the yeast Saccaromyces cerevisiae (for review see Crouse 1997). The MSH2, MSH3,
MSH6, MLH1 and PMS1 proteins appear to function as components of a methyl-directed
pathway similar to that present in E. coli (Table 4). Biochemical studies indicate that MLH1
and PMS1 proteins form a heterodimer called MutLα, which interacts with mismatch-bound
MSH2 during the initiation of MMR in yeast (Prolla et al., 1994). MSH2 can also form a
heterodimer complex with either MSH3 or MSH6 (Marsischky et al., 1996). These
interactions indicate that MSH2 plays a central role in mismatch recognition. The
MSH2/MSH6 complex called MutSα mainly participates in repair of base-base mismatches
(Marsischky et al., 1996), whereas the MSH2/MSH3 complex known as MutSβ, preferentially
binds to 2-4 bp insertions/deletions. Yeast strains deficient in either MSH3 or MSH6 have a
weak mutator phenotype, but yeast strains deficient in both MSH3 and MSH6 have a mutation
rate similar to that observed as a result of MSH2 deficiency. These data suggest that there is a
functional overlap between the MSH3 and MSH6 proteins (Marsischky et al., 1996).
Another likely component of the yeast MMR system is a 5’ to 3’ exonuclease.
Two different candidates have been proposed for this function. One of these whose
involvement in mismatch repair has not been ruled out is the RTH1 (RAD27) gene encoding a
5’ to 3’ exonuclease with a function in DNA replication (Sommers et al., 1995). The second
candidate is EXO1. The EXO1 gene was identified using a two-hybrid system with MSH2 as a
probe (Tishkoff et al., 1997). The EXO1 gene appears to be a homologue of the S. pombe
EXO1 gene, which is a 5’ to 3’ exonuclease involved in mismatch repair in S. pombe. Yeast
strains deficient in EXO1 have increased mutation rates and dinuclotide repeat instability, but
not as high as MSH2 deficient strains. The interaction of EXO1 with MSH2, combined with
the genotype analysis, strongly suggests EXO1 to be a component of the mismatch repair
pathway.
Mismatch repair in humans
The human MMR system is believed to operate in a fashion similar to that of the yeast MMR
system (Fig. 2). The yeast and human MMR proteins show 26-40% similarity in amino acid
sequences (Chung & Rustgi, 1995; Tishkoff et al., 1998; Her & Doggett, 1998). Several
proven or presumed MMR genes have been identified in humans (for reviews see Umar &
Kunkel 1996; Fishel & Wilson 1997). For hMSH2 (an acronym for human MutS Homologue
2 gene), hMSH3 (human MutS Homologue 3), hMSH5 (human MutS Homologue 5), hMSH6
(human MutS Homologue 6), hMLH1 (human MutL Homologue 1), hPMS1 and hPMS2
(human Post Meiotic Segregation, human homologues of MutL genes), and hEXO1 (human
exonuclease homologue 1 gene), some essential characteristics are presented in Table 5.
The MutS homologues
As in yeast, hMSH2 plays a central role in the initial mismatch recognition. Biochemical
studies have shown that hMSH2 forms specific mispair binding complexes with either
hMSH3 or hMSH6 (Acharya et al., 1996; Guerrette et al., 1998). In vitro, the hMSH2 protein
binds
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selectively to DNA containing base-base mismatches and to substrates containing mismatch
loops of up to 14 extra bases (Fishel et al., 1994).
In the majority of homozygous Msh2 knock-out mice early-onset tumourigenesis is
observed (Reitmair et al., 1995; 1996; de Wind et al., 1998). Although one might expect to
find colorectal cancer, in resemblance to human mutation carriers, the predominant tumour is
lymphoma. This type of tumour has been observed in over 80% of the Msh2-/- mice at the age
of one year (Table 6). Lymphomas are relatively rare in HNPCC families (Lynch et al., 1993).
This high incidence of early onset lymphoma may reflect the enhancement of a natural
predisposition to lymphoma in this particular inbred background (Teich et al., 1984). Most
Msh2-/- mice that survived longer than six months also developed intestinal carcinomas. This
proved to be associated with Apc inactivation (Reitmair et al., 1996). In contrast to humans,
where most intestinal carcinomas occur in the proximal colon and rectum, intestinal tumours
of Msh2-/- mice are found mainly in the small intestine. The cause of this tissue specificity is
elusive. In addition, some Msh2-/- mice develop skin tumours, similar to patients of the MuirTorre syndrome which predominantly have hMSH2 mutations (Suspiro et al., 1998; Kruse et
al., 1998). These results support the hypothesis that loss of MMR activity is an important step
in tumourigenesis for several types of cancer that belong to the HNPCC spectrum. As
expected, Msh2 deficiency results in a mutator phenotype. Microsatellite instability is more
common in carcinomas than in adenomas, but virtually absent in normal tissues, suggesting a
causative connection between the appearance of MSI in tumour tissues and defects in MMR
genes. Heterozygous Msh2+/- mice show no overall increase of the incidence of cancer within
the first year of observation (Reitmair et al., 1996). They have not been observed over a longer
periods.
Cell lines that contain mutant hMSH2 genes, exhibit a mutator phenotype (Orth et al.,
1994; Branch et al., 1995). Their mismatch repair activity can be restored by adding a protein
complex, hMutSα, consisting of a heterodimer of hMSH2 and hMSH6 (Drummond et al.,
1995).
As mentioned, the MutS homologue hMSH6 forms a heterodimer complex with hMSH2.
The major function of hMSH6 is obviously to correct base-base mismatches and one or two
base pair deletions/insertions (Fig. 2), since cell lines defective in hMSH6 exhibit elevated
(spontaneous) mutability and are defective in the repair of base-base mismatches and single or
double unpaired bases. However, the degree of instability in hMSH6-defective cells is not as
high as in hMSH2-defective cells (Shibata et al., 1994; Papadopoulos et al., 1995a;
Bhattacharyya et al., 1995). Extracts of hMSH6-defective cells are partially proficient in
repairing substrates containing 2-4 unpaired bases (Drummond et al., 1995). Therefore, the
repair of heteroduplexes might be initiated by hMSH2 alone or complexed with another MMR
protein.
Msh6-/- mice have a significantly reduced life span and develop a spectrum of tumours
(Edelmann et al., 1997). Most predominant are gastrointestinal tumours and B- as well as Tcell lymphomas (Table 6). All these tumours show little or no MSI. Extracts of Msh6-/- cells
are defective for repair of base-base mismatches, but repair of 1, 2, and 4 nucleotide
insertions/deletions is unaffected (Edelmann et al., 1997). This is consistent with a model in
which MSH6 is involved in correction of base-base mismatches (Drummond et al., 1995).
Although Msh6-/- mice have a significantly reduced life span it is longer than that of Msh2-/mice. Msh6-/- mice also develop tumours somewhat later in life. This may suggest a small
compensatory role for other MMR genes, such as Msh3.
Mutation in hMSH3, the third human MutS homologue gene (Shinya & Shimada, 1994),
seems to have little effect on the (spontaneous) base-base mismatches at several loci, but does
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strongly elevate the mutation rate for insertions/deletions in dinucleotide sequences (Strand et
al., 1995), suggesting that hMSH3 would bind hMSH2 for the repair of small
deletions/insertions (Fig. 2).
Another human MutS homologue gene, hMSH5, has recently been identified (Her et al.,
1998). Msh5-/- mice are viable but sterile (Edelmann et al., 1999). Meiosis in these mice is
affected due to the disruption of chromosome pairing in prophase I. Msh5-/- knock-out mice
show that normal Msh5 function is essential for meiotic progression and, in females, gonadal
maintenance.
The MutL homologues
The hMLH1, hPMS1 and hPMS2 genes all share sequence similarity to the bacterial MutL
gene. The hPMS1 and hPMS2 genes are related to the yeast MLH2 and PMS1 genes,
respectively (Table 4). Cells containing mutations in hMLH1 or hPMS2 have elevated rates of
spontaneous mutation (for review see Umar and Kunkel, 1996). Repair activity of a hMLH1defective tumour cell line can be restored by a protein fraction designated hMLH1α (Li et al.,
1995), consisting of a heterodimer of hMLH1 and a second protein, most likely hPMS2 (Fig.
2). Thus, at least two gene products are required in yeast and human cells to fulfil the function
of one MutL in E. coli.
Mlh1-/- mice usually develop lymphomas, intestinal tumours associated with Apc
inactivation, and to a lesser extent skin tumours and sarcomas (Baker et al., 1996, Prolla et
al., 1998) (Table 6). As expected, Mlh1-deficient tumours display high levels of MSI. The
pattern of tumour development and the MSI phenotype are similar to those of Msh2-/- mice
(Reitmair et al., 1996). These data suggest that Msh2 and Mlh1 are both very important in the
MMR pathway. Mlh1-/- mice are viable, but both males and females are sterile due to the
premature separation of paired homologous chromosomes which causes arrest in the first
division of meiosis (Baker et al., 1996; Edelman et al., 1996).
Mice deficient for the Pms1 gene do not develop any tumours (Prolla et al., 1998) (Table
6). MSI in the colonic mucosa of Pms1-/-mice is rare and not significantly higher than in the
mucosa of wild-type mice. In mouse embryonic fibroblast cells PMS1 deficiency has no effect
on mutations in dinucleotide repeats, but a significant effect on mutations in mononucleotide
repeats. Pms1-/- mice are viable and, unlike Mlh1 and Pms2 deficient mice, fertile. Possibly,
the PMS1 protein plays a role in a novel MMR pathway which is independent of MLH1 and
PMS2.
Pms2-/- mice develop either lymphomas or sarcomas (Prolla et al., 1998), T-cell lymphomas
being most predominant (Table 6). Different from mice deficient in the other MutL
homologues, such as Mlh1-/- mice, none of the Pms2-/- mice developed intestinal adenomas or
adenocarcinomas. These findings suggest distinct roles for the Mlh1 and Pms2 genes in
tumour development. Established mouse embryonic fibroblast cells deficient in either Mlh1 or
Pms2 show frequent alterations in both mononcleotide and dinucleotide repeats. Pms2-/- mice
display female sterility (Baker et al., 1995).
The MutH homologues
Human MutH homologue genes have not (yet) been identified.
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The EXO1 homologue
Recently, a human exonuclease (hEXO1) was cloned homologous to yeast exonuclease 1. The
hEXO1 was found to interact strongly with the human MMR protein hMSH2, suggesting its
involvement in the MMR process and/or DNA recombination (Schmutte et al., 1998b).
Other roles for mismatch repair proteins
Besides correction of replication errors, mismatch repair proteins may also have other
functions (for review see Umar and Kunkel, 1996). The MMR system is presumed to mediate
toxicity of methylating agents (Koi et al., 1994). It may, as indicated above, also be involved
in the prevention of recombination between homologous nonidentical DNA sequences during
meiosis (Baker et al., 1996; Edelmann et al., 1996). Therefore, failure of the MMR system
may result in tolerance to the cytoxic effects of methylating agents and in enhanced
recombination between diverged DNA sequences.
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