Functional Overlap in Mismatch Repair by Human MSH3 and MSH6

Corresponding author: Thomas A. Kunkel, Laboratory of Molecular Genetics, E3-01, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, kunkel{at}niehs.nih.gov (E-mail).

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Three human genes, hMSH2, hMSH3, and hMSH6, are homologues of the bacterial MutS gene whose products bind DNA mismatches to initiate strand-specific repair of DNA replication errors. Several studies suggest that a complex of hMSH2·hMSH6 (hMutS) functions primarily in repair of base·base mismatches or single extra bases, whereas a hMSH2·hMSH3 complex (hMutSß) functions chiefly in repair of heteroduplexes containing two to four extra bases. In the present study, we compare results with a tumor cell line (HHUA) that is mutant in both hMSH3 and hMSH6 to results with derivative clones containing either wild-type hMSH3 or wild-type hMSH6, introduced by microcell-mediated transfer of chromosome 5 or 2, respectively. HHUA cells exhibit marked instability at 12 different microsatellite loci composed of repeat units of 1 to 4 base pairs. Compared to normal cells, HHUA cells have mutation rates at the HPRT locus that are elevated 500-fold for base substitutions and 2400-fold for single-base frameshifts. Extracts of HHUA cells are defective in strand-specific repair of substrates containing base·base mismatches or 1–4 extra bases. Transfer of either chromosome 5 (hMSH3) or 2 (hMSH6) into HHUA cells partially corrects instability at the microsatellite loci and also the substitution and frameshift mutator phenotypes at the HPRT locus. Extracts of these lines can repair some, but not all, heteroduplexes. The combined mutation rate and mismatch repair specificity data suggest that both hMSH3 and hMSH6 can independently participate in repair of replication errors containing base·base mismatches or 1–4 extra bases. Thus, these two gene products share redundant roles in controlling mutation rates in human cells.

THE pace of studies to identify and determine the functions of DNA mismatch repair (MMR) genes in humans accelerated when mutations in the hMSH2 gene were first linked to hereditary colon cancer (FISHEL et al. 1993 ; LEACH et al. 1993 ). This gene is one of several (reviewed in FISHEL and WILSON 1997 ) that share sequence homology with the bacterial mismatch repair gene MutS, whose product binds to mismatches and initiates their repair. Human MSH2 is located on the short arm of chromosome 2 and encodes a protein of 105 kD. The human (FISHEL et al. 1994A , FISHEL et al. 1994B ) and yeast (ALANI et al. 1995 ) MSH2 proteins bind to DNA containing base·base mispairs and to substrates containing from one to 14 extra bases. Tumor cell lines with mutant hMSH2 genes have elevated spontaneous mutation rates in endogenous genes (BRANCH et al. 1995 ; MALKHOSYAN et al. 1996 ; RICHARDS et al. 1997 ; UMAR et al. 1997 ) and exhibit microsatellite instability (ORTH et al. 1994 ; UMAR et al. 1994A ; BOYER et al. 1995 ; RISINGER et al. 1995 ; SHIBATA et al. 1995 ). Similarly, msh2 mutant yeast strains also have strongly elevated mutation rates (see below and, for review, see SIA et al. 1997B ). Studies in vitro reveal that extracts of hMSH2-mutant cells are defective in the strand-specific repair of substrates containing mismatches or extra bases (UMAR et al. 1994A , UMAR et al. 1994B , UMAR et al. 1997 ; BOYER et al. 1995 ; DRUMMOND et al. 1995 ; LI et al. 1995; RISINGER et al. 1995 ). hMSH2-mutant cells are also more resistant to killing by certain DNA-damaging agents than are MMR-proficient cells (RISINGER et al. 1995 ; UMAR et al. 1997 ). Transfer of chromosome 2 from a repair-proficient cell to an hMSH2-mutant cell lowers the mutation rate, stabilizes microsatellites, and sensitizes the cells to MNNG (UMAR et al. 1997 ) and cisplatin (FINK et al. 1996 ). MMR activity is restored to extracts of hMSH2-mutant cells both by chromosome 2 transfer (UMAR et al. 1997 ) and by addition of the hMutS protein complex (DRUMMOND et al. 1995 ). hMutS is a heterodimer of hMSH2 and the 160-kD product of the gene located on chromosome 2p that is homologous to the yeast MSH6 gene.¹ Efficient binding of hMutS to a G ·T mismatch requires both hMSH2 and hMSH6 proteins (PALOMBO et al. 1995 ), and yeast MutS likewise binds to this mismatch (IACCARINO et al. 1996 ). Human and yeast MutS bind to substrates containing extra nucleotides in one strand (DRUMMOND et al. 1995 ; ALANI 1996 ; PALOMBO et al. 1996 ; ALANI et al. 1997 ) and to a variety of damaged DNA substrates (DUCKETT et al. 1996 ; LI et al. 1996 ; MELLO et al. 1996 ; MU et al. 1997 ).

These data strongly suggest that MSH2 is an essential protein for the initial mismatch recognition step in the strand-specific repair of replication errors in eukaryotic cells (for review, see JIRICNY 1996 ; KOLODNER 1996 ; MODRICH and LAHUE 1996 ; UMAR and KUNKEL 1996 ). The presence of hMSH6 in the hMutS complex suggests that it too functions in mismatch repair. In support of this hypothesis, hMSH6-mutant human tumor cells are mutators (BHATTACHARYYA et al. 1994 ; BRANCH et al. 1995 ; MALKHOSYAN et al. 1996 ; GLAAB and TINDALL 1997 ; OHZEKI et al. 1997 ) and are resistant to killing by alkylating agents (KAT et al. 1993 ; BRANCH et al. 1995 ; UMAR et al. 1997 ) and 6-TG (W. E. GLAAB, J. I. RISINGER, A. UMAR, J. C. BARRETT, T. A. KUNKEL, unpublished results). Extracts of these cells are also deficient in MMR activity (KAT et al. 1993 ; UMAR et al. 1994A , UMAR et al. 1997 ; DRUMMOND et al. 1995 ; RISINGER et al. 1996 ). All three of these phenotypes are reversed by transferring chromosome 2 from a repair-proficient cell into an hMSH6-mutant cell (UMAR et al. 1997 ; W. E. GLAAB, J. I. RISINGER, A. UMAR, J. C. BARRETT, T. A. KUNKEL and K. R. TINDALL, unpublished results). However, the degree of microsatellite instability in hMSH6-mutant cells is not as high as in hMSH2-mutant cells (BHATTACHARYYA et al. 1995 ; PAPADOPOULOS et al. 1995 ; SHIBATA et al. 1995 ). Also, studies in yeast (STRAND et al. 1993 ; JOHNSON et al. 1996 ; MARSISCHKY et al. 1996 ; SIA et al. 1997A ) clearly show that, although a null mutation in MSH2 strongly elevates the rate of a variety of mutations, a null mutation in MSH6 has a smaller effect on the rate of some frameshift mutations and a negligible effect on others. Moreover, extracts of MSH6-mutant human tumor cells are only partially repair-deficient. Although they fail to repair single base mismatches, they do retain the ability to repair some insertion/deletion mismatches (DRUMMOND et al. 1995 ; RISINGER et al. 1996 ; UMAR et al. 1997 ). Thus, repair of some mismatches may be initiated by hMSH2 alone or in complex with another protein.

A candidate for this other protein is MSH3, a third eukaryotic MutS homologue. Yeast strains containing a null mutation in MSH3 are mutators ( JOHNSON et al. 1996 ; MARSISCHKY et al. 1996 ; SIA et al. 1997A ), indicating a role for this gene in MMR. Several studies of yeast strains containing MSH2, MSH3, and MSH6 mutations (STRAND et al. 1995 ; JOHNSON et al. 1996 ; MARSISCHKY et al. 1996 ; GREENE and JINKS-ROBERTSON 1997 ; SIA et al. 1997A ) suggest that MSH2 and MSH6 are primarily responsible for repairing base·base mismatches. MSH2 can participate with either MSH6 or MSH3 to repair one- and two-base insertion/deletion mispairs, whereas repair of insertion/deletions involving repeating units of four to 16 bases requires MSH2 and MSH3 but not MSH6. Yeast MSH2 protein has been shown to physically interact with both MSH6 and MSH3 proteins translated in vitro (MARSISCHKY et al. 1996 ). Recombinant yeast MSH2 and MSH3 proteins form a stable heterodimer that has low affinity for binding to a G ·T mismatch but has higher binding affinity for insertion/deletion mismatches involving 2–14 extra bases (HABRAKEN et al. 1996 ). These data support the hypothesis that at least two different MSH2-dependent, strand-specific MMR processes exist in yeast.

Evidence that MSH3 functions in strand-specific MMR in human cells comes from studies of a human endometrial tumor cell line, HHUA (RISINGER et al. 1996 ). The hMSH3 gene is on chromosome 5 and encodes a protein of 1137 amino acids (reviewed in FISHEL and WILSON 1997 ). HHUA cells harbor a single base deletion mutation in hMSH3 and the MSH3 protein is missing 723 C-terminal amino acids, many of which are conserved among eukaryotic MutS homologues. Microsatellites are highly unstable in HHUA cells, and an extract of HHUA cells is unable to repair base·base or insertion/deletion mismatches (RISINGER et al. 1996 ). Furthermore, transfer of chromosome 5 from a repair-proficient cell to HHUA cells stabilizes microsatellites, and extracts of these cells exhibit strand-specific repair of one- and four-base deletion mismatches (RISINGER et al. 1996 ). Consistent with these results, recombinant human MSH2 and MSH3 form a stable heterodimer that binds to one- to four-base insertion/deletion mismatches (PALOMBO et al. 1996 ). Collectively, the data strongly suggest a functional role for human hMSH3 in MMR repair.

In our initial study (RISINGER et al. 1996 ), HHUA cells containing wild-type chromosome 5 still exhibited a detectable level of microsatellite instability, and extracts of these cells still lacked MMR activity for base·base mismatches and some insertion mismatches. These results suggested that HHUA cells were mutant in another mismatch repair gene. Subsequent analysis revealed a mutation in hMSH6 that was consistent with loss of function (see below). The fact that HHUA cells are mutant in both hMSH3 and hMSH6 provides the opportunity to investigate the roles of these two genes in human cells by studying each in the absence of the other. To do so, we have introduced chromosome 2 into HHUA cells to allow direct comparison of a hMSH3/hMSH6 double mutant (HHUA) to derivatives corrected for one or the other MMR gene (HHUA plus chromosome 2 or chromosome 5 ). Here we describe three properties of these three cell lines: microsatellite instability and HPRT gene mutation rates in vivo and MMR capacity in vitro. The results indicate that hMSH3 and hMSH6 share redundant roles in human cells.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cell lines, analysis of microsatellite instability, and sequence analysis of hMSH6:
The source and growth of cell lines is given in RISINGER et al. 1996 . The method used to analyze microsatellite stability in single cell clones has been described (RISINGER et al. 1996 ). Sequence analysis of hMSH6 was performed as described previously (RISINGER et al. 1996 ), using primers described in PAPADOPOULOS et al. 1995 .

Chromosome transfer:
The method used for chromosome transfer is described in KOI et al. 1989 . The introduced chromosomes contain the gene conferring resistance to G418, such that cells receiving that chromosome are resistant to this drug. The presence of a transferred chromosome 2 was confirmed by detecting the allele of the donor cell line in early passage DNAs using the tetranucleotide repeats D2S405, D2S407, D2S410, D2S423, and D2S433. When genomic DNAs from the chromosome-transferred cell lines described were amplified using PCR primers specific for mouse B2 repetitive sequence elements, no detectable mouse DNA was observed.

HPRT mutation frequency and rate determinations:
Mutation frequencies were obtained by plating 10⁶ cells in 40 µmol 6-TG at a density of 5 x 10⁴ cells per 10-cm dish. Cells were incubated 12–14 days and 6-TG^r colonies were visualized by staining with 0.5% crystal violet (in 50% methanol, v/v). Mutation rate determinations were performed using cell populations cleansed of preexisting HPRT mutants by culture in HAT (100 µmol hypoxanthine, 0.4 µmol aminopterin, and 16 µmol thymidine) medium. HAT medium was then removed and the initial HPRT mutant frequency was determined, and 2–3 x 10⁶ cells were subcultured in nonselective medium. Additional mutant frequencies were obtained at 2- to 3-day intervals, while maintaining the cells in logarithmic growth. At each 6-TG selection, a subculture of 2–3 x 10⁶ cells was plated in nonselective medium for use in subsequent mutant frequency determinations. Additionally, population doublings were determined between each 6-TG selection. After obtaining 5–6 mutant frequencies, mutation rates were obtained by plotting the observed mutant frequency as a function of population doubling and calculating the slope by linear regression. The slope of the curve yields the mutation rate, expressed as mutations per cell per generation (GLAAB and TINDALL 1997 ).

HPRT mutant selection and sequencing:
Independent mutants resistant to 6-TG were obtained by first cleansing cell cultures of preexisting HPRT mutants in HAT medium. Following removal of HAT medium, 100 cells were plated in nonselective medium. These independent 100-cell cultures were grown to ~2.5 x 10⁶ cells and then selected in 6-TG as described above. Colonies were allowed to grow for 14–18 days, and individual 6-TG^r clones were isolated. These 6-TG^r clones were transferred to 24-well dishes and grown to confluence. Independent mutants were defined as those spontaneous mutants arising in different 100-cell inocula. Amplification of HPRT mRNA from mutant clones was then performed by a procedure modified from YANG et al. 1989 . The cDNA of the HPRT gene was then analyzed by automated sequencing.

Assay for mismatch repair activity:
A circular M13mp2 DNA substrate is used, containing a covalently closed (+) strand and a (-) strand with a nick (to direct repair to this strand) located several hundred base pairs away from the mispair located in the lacZ -complementation coding sequence. The (+) strand encodes one plaque phenotype (either colorless or blue) and the (-) strand encodes the other plaque phenotype. When the unrepaired heteroduplex is introduced into an Escherichia coli strain deficient in methyl-directed heteroduplex repair, plaques have a mixed plaque phenotype on selective plates, because both strands of the heteroduplex are expressed. However, repair occurring during incubation of the substrate in a repair-proficient human cell extract will reduce the percentage of mixed plaques and increase the ratio of the (+) strand phenotype relative to that of the (-) strand phenotype, because the nick directs repair to the (-) strand. The method for preparing extracts for repair reactions is described in ROBERTS and KUNKEL 1993 . Preparation of mismatched substrates and procedures for measuring mismatch repair activity have been described in THOMAS et al. 1995 . Repair reactions (25 µl) contained 30 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (pH 7.8), 7 mM MgCl₂, 200 µmol each CTP, GTP, UTP, 4 mM ATP, 100 µmol each dCTP, dATP, dGTP, dTTP, 40 mM creatine phosphate, 100 mg/ml creatine phosphokinase, 15 mM sodium phosphate (pH 7.5), 1 fmol of substrate DNA, and 50 µg of extract proteins. Following incubation at 37° for 30 min, the DNA substrates were processed and introduced into E. coli NR9162 (mutS) via electroporation. Cells were plated, plaque colors were scored, and repair efficiency calculated as described (THOMAS et al. 1995 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Introduction of hMSH6 into HHUA cells:
HHUA cells contain a single base deletion mutation in hMSH3, such that the MSH3 protein is missing 723 C-terminal amino acids (Figure 1A). Many of these residues are conserved among eukaryotic MutS homologues, consistent with the loss of hMSH3 function observed in an earlier study (RISINGER et al. 1996 ). However, HHUA cells containing wild-type chromosome 5 still exhibited a detectable level of microsatellite instability, and extracts of these cells still lacked MMR activity for base ·base mismatches and some insertion mismatches. These results suggested that HHUA cells were mutant in another mismatch repair gene. Subsequent analysis of hMSH6 (RISINGER et al. 1996 ) revealed a homozygous CT mutation (Figure 1B) that changed codon 1219 from threonine (ACT) to isoleucine (ATT). Threonine is present at this position in 12 of 14 MutS homologues (Figure 1C), suggesting that the change to isoleucine may impair hMSH6 function.

View larger version (21K): In this window In a new window Download PPT slide   Figure 1. —Mutations in hMSH3 and hMSH6 in HHUA cells. (A) A schematic drawing of hMSH3 and corresponding mutation in HHUA cell line. The total number of amino acid residues is indicated in parentheses. Shaded areas represent conserved regions of the protein. The position of highly conserved Walker type A nucleotide binding sequence (GKS) is indicated as a dark bar. A indicates the loss af an A·T base pair, resulting in protein truncation. (B) A schematic drawing of hMSH6 and the corresponding mutation in the HHUA cell line. The total number of amino acid residues is indicated in parentheses. Shaded areas represent conserved regions of the protein. The position of highly conserved Walker type A nucleotide binding sequence (GKS) is indicated as a dark bar. (C) Alignment of the conserved region of MutS homologues at the region where the mutation in HHUA cells is found in hMSH6 protein. The top line represents the consensus sequence; identical sequences are indicated by a (·) and differences from consensus for each MutS homologue are indicated by the single letter code for amino acids. The bottom line shows the sequence for hMSH6 in the HHUA cell line. Eco, E. coli; Spell 1, Spellchecker 1 (the MutS homologue in Drosophila melanogaster); x, Xenopus ; y, Saccharomyces cerevisiae ; m, mouse; r, rat; h, human.

We began this study by transferring chromosome 2 (encoding hMSH6 ²) into HHUA cells by microcell fusion (KOI et al. 1989 ). Single cell clones receiving chromosome 2 were selected for resistance to G418, and the presence of the wild-type hMSH6 gene sequence in G418-resistant clones was analyzed by DNA sequence analysis of the region of hMSH6 known to be mutant in HHUA cells. Both the HHUA parent cells (left sequence in Figure 2) and a clone (HHUA-5.5) containing chromosome 5 (middle sequence in Figure 2) had only the mutant ATT sequence at codon 1219 reported earlier (RISINGER et al. 1996 ). In contrast, a clone receiving chromosome 2 contained both the mutant ATT codon and a wild-type ACT codon 1219.

View larger version (79K): In this window In a new window Download PPT slide   Figure 2. —MSH6 gene sequence in HHUA and its derivatives. The sequence analysis was performed as described in materials and methods.

Analysis of microsatellite instability:
As is typical of many cell lines with mutations in mismatch repair genes, hMSH3/hMSH6-mutant HHUA cells exhibited microsatellite instability (RISINGER et al. 1996 ). This includes the parental HHUA clone used here, where, for any given microsatellite, instability was observed in from four to 19 of the 36 single cell clones examined (Table 1, parent line). Instability was also observed in from five to 11 of 36 clones derived from a clone (17.3 in Table 1) into which chromosome 17 was introduced, used here as a negative control. In all, 12 microsatellite loci were examined, including four homopolymeric sequence loci, five loci comprised of dinucleotide repeats, two trinucleotide repeat loci, and one tetranucleotide microsatellite. The instabilities observed included both additions and deletions of one or more repeat units of one to four bases.

The extent of microsatellite instability was reduced in single cell clones derived from two clones (2.5 and 2.10 in Table 1) into which chromosome 2 (hMSH6) was introduced. For example, although 19 of 36 parental HHUA clones were unstable at the D17S791 dinucleotide repeat locus, none of 24 clones of the 2.5 derivative and only two of 36 clones of the 2.10 derivative were observed to be unstable. Reduced instability was observed at all loci examined (Table 1). These results suggest that the human MSH6 gene participates in the repair of addition and deletion replication errors at all of these loci. This includes errors in which the inferred mutational intermediates could involve 1, 2, 3, or 4 extra bases residing in the primer strand (additions) or the template strand (deletions). Single cell clones derived from HHUA cells into which chromosome 5 (hMSH3) was introduced (5.5 and 5.10 in Table 1) also exhibited reduced instability. Thus, among 71 total clones examined (35 for the 5.5 derivative and 36 for the 5.10 derivative), few loci were observed to be unstable. However, the stabilizing effects of chromosome 5 transfer were confined to di-, tri-, and tetranucleotide microsatellites. Increased stability was not apparent for the four homopolymeric microsatellites examined in clone 5.5. These data suggest that the human MSH3 gene also participates in the repair of addition and deletion replication errors involving 2, 3, or 4 extra bases. However, there is no indication of hMSH3-dependent repair of single-base deletions at the four homopolymeric loci examined.

Analysis of mutation rates at the HPRT locus:
To explore further the mutator phenotype of HHUA cells and the effects of introduction of chromosomes 2 and 5, we determined mutation rates at the HPRT locus in the HHUA parent clone and its derivatives. Consistent with results obtained with hMSH2 and hMSH6 single-mutant tumor cell lines (BHATTACHARYYA et al. 1994 ; BRANCH et al. 1995 ; MALKHOSYAN et al. 1996 ; W. E. GLAAB, J. I. RISINGER, A. UMAR, J. C. BARRETT, T. A. KUNKEL and K. R. TINDALL, unpublished results; OHZEKI et al. 1997 ), the double-mutant HHUA parent clone had an elevated HPRT mutation rate of 2.1 x 10^-5, or 450-fold higher than the rate for normal human fibroblasts (Table 2). Clone 17.3, which showed no reduction in microsatellite instability (Table 1), also had a high mutation rate (1.7 x 10^-5). The rate for HHUA cells into which the hMSH6 gene on chromosome 2 was introduced (clone 2.10) was 2.9 x 10^-7 (Table 2). This value is 72-fold lower than that of the double-mutant HHUA cells, indicating a functional role for the hMSH6 gene. HHUA cells into which the hMSH3 gene on chromosome 5 was introduced (clone 5.10) also had a lower mutation rate (3.2 x 10^-6; Table 2). This value is 6.6-fold lower than the rate for HHUA cells, indicating that the hMSH3 gene also functions in MMR. However, the mutation rate in clone 5.10 remains 68-fold higher than that of normal human fibroblasts (Table 2) known to be MMR proficient (BOYER et al. 1993 ). Thus, introduction of hMSH3 yields only partial correction of the mutation rate.

The specificity of spontaneous mutagenesis in mismatch repair-proficient human cells is known, allowing calculation of the rates for single-base substitution and frameshift mutations at the HPRT locus (Table 2). Spontaneous mutations in HHUA included 57% single-base substitutions (25 of 44 independent mutants) and 43% single-base frameshifts (19 of 44 independent mutants), 11 of which were in a homopolymeric run of six guanines (base 207–212) in exon 3. The calculated mutation rates (Table 2) reveal that the overall base substitution rate in HHUA cells was elevated 500-fold relative to normal fibroblasts, with the rate of transition mutations elevated to a twofold greater extent than that of the transversion rate. These hMSH3/hMSH6 - mutant cells have an even more strongly elevated rate of single-base frameshifts, which are increased by 2400-fold relative to NHF-1 cells. There is an approximately fourfold greater increase in rate observed for additions than for deletions.

Using the overall low HPRT mutation rate of 2.9 x 10^-7 in HHUA clone 2.10 cells, we calculated the minimum extent to which introduction of chromosome 2 containing the hMSH6 gene lowered the mutation rate relative to parental HHUA cells. The results (Table 2) show that the rates for transitions, transversions, additions, and deletions were reduced by 96, 91, 92, and 94%, respectively. The spectrum of spontaneous mutations observed in clone 5.10 was similar to that observed in the parental HHUA cells. There were slightly more base substitutions (six of nine independent mutants) than frameshift mutations (three of nine independent mutants). Calculations based on these data (Table 2) show that the rates for transitions, transversions, additions, and deletions were reduced by 90, 67, 81, and 92%, respectively, in cells receiving chromosome 5 encoding the hMSH3 gene.

Restoration of MMR activity in extracts:
Extracts of HHUA cells were deficient in strand-specific mismatch repair in vitro of substrates containing base·base and insertion/deletion mismatches. As previously reported (RISINGER et al. 1996 ), extracts of clone 5.5 cells repaired some substrates containing extra nucleotides but were deficient in repair of those containing base·base mismatches or certain other extra nucleotides. To determine the effect on MMR activity of introducing chromosome 2 into HHUA cells, we examined the ability of extracts of the clones receiving chromosome 2 (hMSH6) to repair nine heteroduplex substrates in vitro. An extract of MMR-proficient HeLa cells repaired all of these substrates in a strand-specific manner, whereas an extract of parental HHUA cells failed to repair any of them (Figure 3). Extracts of HHUA clone 2.5 cells repaired seven of the nine substrates. Of the two substrates that were not repaired, one contained a single extra nucleotide and the other contained two extra bases (fourth and fifth substrates shown in Figure 3). Note that these substrates are repaired in an extract of MMR-proficient HeLa cells. The seven substrates that were repaired in extracts of HHUA clone 2.5 cells included a G·G and a G·T mismatch and heteroduplexes containing one to four extra bases in one strand (Figure 3). In all cases where repair was observed, the change in the ratio of blue to colorless plaques indicates that repair occurred in the strand containing the nick, which is known to serve as a strand-discrimination signal in vitro (HOLMES et al. 1990 ; THOMAS et al. 1991 ). Repair was observed when the nick was either 3' or 5' to the mismatch, indicating that this repair activity that depends on the newly introduced chromosome 2 has the characteristic bidirectional repair capacity of the general MMR system (FANG and MODRICH 1993 ). Thus, the data in Figure 3 demonstrate that introduction of chromosome 2 into HHUA cells restored to extracts the ability to perform strand-specific MMR to a variety of heteroduplexes.

View larger version (34K): In this window In a new window Download PPT slide   Figure 3. —Mismatch repair activity in HHUA and its derivative cell lines. The analysis was performed as described in materials and methods using the substrates indicated (described in RISINGER et al. 1996 ) and reactions were incubated for 15 min. Substrates designated with a () contain the number of extra nucleotides that accompany the symbol. Substrates are designated with a 3' when the nick in the (-) strand is at the AvaII site (position -264, where position +1 is the first transcribed base of the lacZ -complementation gene) or with a 5' when the nick is at the Bsu36 I site (position +276). The nucleotide position of the mismatch or unpaired bases in the lacZ -complementation gene is indicated after the symbol @. In all substrates, the nick is in the (-) strand. The (+) or (-) sign designates the strand containing the extra base(s). Results are expressed as percent repair determined from counting several hundred plaques per variable. Repair values of 1% are represented as 1%. All three extracts were competent for SV40 origin-dependent DNA replication activity.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The fact that introduction of chromosome 2 (hMSH6) from an MMR-proficient cell into the hMSH3/hMSH6-mutant, MMR-deficient HHUA human endometrial tumor cell line reduces microsatellite instability at 12 loci (Table 1), reduces the mutation rate at the HPRT locus (Table 2), and restores MMR activity to extracts in vitro (Figure 3) suggests a functional role for the hMSH6 gene in postreplication mismatch repair. The data are consistent with many other observations in yeast and human cells, including the fact that introduction of chromosome 2 also corrects an independently derived colon tumor cell line containing other hMSH6 mutations (UMAR et al. 1997 ). A functional role in MMR is inferred for hMSH3 as well, on the basis of the fact that introduction of chromosome 5 encoding the hMSH3 gene into hMSH3/hMSH6-mutant HHUA cells also reduces microsatellite instability and restores MMR activity (RISINGER et al. 1996 ). That earlier study of hMSH3 gene function is extended here by examination of several additional microsatellite loci (Table 1) and by the demonstration that introducing chromosome 5 also reduces the HPRT mutation rate (Table 2).

Human tumor cell lines with mutations in hMSH2 exhibit robust microsatellite instability, highly elevated mutation rates in endogenous genes, and deficiency in repair of a variety of base·base and insertion/deletion mismatches (see citations in Introduction). These data and studies (cited above) of the substrate binding specificity of MSH2·MSH6 and of MSH2·MSH3 heterodimers suggest that MSH2 is involved in the initial recognition step of strand-specific repair of a wide variety of replication errors in eukaryotic cells. The hMSH3^-/hMSH6^- double mutant HHUA cell line has similar genetic instability and MMR deficiencies as MSH2-mutant cells, and partial correction is observed upon introduction of either wild-type hMSH3 or hMSH6, suggesting that hMSH3 and hMSH6 share partially redundant functions in MMR in human cells. Table 3 summarizes the inferred extent of redundancy in human cells and compares the results to those obtained in yeast.

With respect to MSH6 function, the reduced substitution rate at the HPRT locus (Table 2) and the ability to repair base·base mismatches in vitro (Figure 3) upon introduction of chromosome 2 into HHUA cells implies that hMSH6 participates in repair of base·base mismatches. This is consistent with the increased base substitution rate at the canavanine locus in a yeast msh6 mutant (MARSISCHKY et al. 1996 ) and with the ability of human and yeast MutS to bind to base·base mismatches (cited above). Introduction of chromosome 2 into HHUA cells results in increased stability of microsatellites comprised of one-, two-, three-, or four-base repeats (Table 1), a reduction in mutation rates for single-base deletions and additions at the HPRT locus (Table 2), and an ability of extracts to repair some (but not all) substrates containing one, two, three, or four extra bases (Figure 3). This implies that human MSH6 also participates in the repair of insertion/deletion mismatches involving one, two, three, or four extra bases. Mononucleotide and dinucleotide repeats are likewise unstable in a yeast msh6 mutant (Table 3), consistent with MSH6-dependent repair of insertion/deletion mismatches containing one or two extra bases. However, SIA et al. 1997A observed no effect of an msh6 mutation on the stability of repetitive sequences containing repeat units of four or more bases. Thus, MSH6-dependent repair specificity could possibly differ in yeast and human cells. Alternatively, the difference could reflect the use of different repeat sequences or any of several other variables (discussed below).

With respect to MSH3 function, introduction of chromosome 5 (hMSH3) into HHUA cells reduced the base substitution rate at the HPRT locus (Table 2). This implies that hMSH3 can participate in repair of base·base mismatches, at least under circumstances where hMSH6 is mutant. A limited role for MSH3 in repair of base·base mismatches is consistent with the slight but detectable binding of human (Figure 4a in PALOMBO et al. 1996 ) and yeast (Figure 3B in HABRAKEN et al. 1996 ) MSH2·MSH3 complexes to a G·T mismatch. Moreover, binding of the yeast MSH2·MSH3 complex is enhanced by the yeast MLH1·PMS1 complex (HABRAKEN et al. 1997 ). A lower apparent affinity of MSH2·MSH3 for base·base mispairs as compared to MSH2·MSH6 may explain why the effect on base substitution rates at the HPRT locus was less for introduction of chromosome 5 than for introduction of chromosome 2 into HHUA cells (Table 2). A lower binding affinity of MSH2·MSH3 to base·base mismatches may also explain why introduction of chromosome 5 failed to restore base·base mismatch repair activity to extracts (Table 3; RISINGER et al. 1996 ). It is also noteworthy that the degree to which the base substitution rate at the canavanine locus in yeast is elevated in msh2, msh3, and msh6 mutants suggests that MSH3 may not participate in base·base mismatch repair in yeast (MARSISCHKY et al. 1996 ), again providing at least the possibility of differences between the yeast and human mismatch repair systems.

Introducing hMSH3 on chromosome 5 into HHUA cells increases the stability of microsatellites comprised of two-, three-, or four-base repeats (Table 1), decreases the rate of single-base deletions and additions at the HPRT locus (Table 2), and restores to extracts the capacity to repair some (but not all) substrates containing one or four extra bases (RISINGER et al. 1996 ). This implies that human MSH3 also participates in repairing some insertion/deletion mismatches involving one, two, three, or four extra bases. This conclusion is consistent with five studies in mutant yeast strains (Table 3).

These inferences on the role of MSH3 and MSH6 in repair of various types of mismatches are derived from studies in which one gene is mutant and the other wild type. Unknown is the relative contribution of MSH3 and MSH6 to repair of different mismatches in normal cells containing two wild-type copies of both genes. Further studies will be required to understand why, despite the apparent ability to repair some insertion/deletion mismatches, extracts of HHUA-2.5 cells (Figure 3) and HHUA-5.5 cells (Figure 3 in RISINGER et al. 1996 ) fail to repair certain others and why, even after introducing chromosome 5, HHUA cells continue to exhibit instability at four homopolymeric microsatellites (Table 2). These data indicate that MSH6- and MSH3-dependent insertion/deletion mismatch repair capacity may depend on variables other than simply the number of extra bases. These variables may include the identity of the extra nucleotides, whether they are present in the template strand (as for deletions) or the primer strand (as for additions) or within repetitive or nonrepetitive sequences. Also, repair capacity may be influenced by the location of the mismatch relative to the (presently unknown) strand-discrimination signal, relative to the replication fork (e.g., leading or lagging strand replication errors), or relative to transcription. The cell lines described here also provide the opportunity to examine the effects of mutations in hMSH3 and hMSH6 on induced cytotoxicity and mutagenesis (e.g., see W. E. GLAAB, J. I. RISINGER, A. UMAR, J. C. BARRETT, T. A. KUNKEL and K. R. TINDALL, unpublished results).

	FOOTNOTES

¹ The human gene is also known as p160 (DRUMMOND et al. 1995 ) and GTBP (PALOMBO et al. 1995 ; PAPADOPOLOUS et al. 1995), the latter for G ·T mismatch binding protein, the property upon which the gene product was first identified (HUGHES and JIRICNY 1992 ). For simplicity and given its homology to the yeast MSH6 gene, we refer to the human gene as hMSH6 and its gene product as hMSH6 protein.
² For clarity, we have in several places provided the name of the relevant MutS homologue in parentheses after the chromosome that was transferred.

	ACKNOWLEDGMENTS

We thank SAMUEL E. BENNETT and KARIN DROTSCHMANN for critically evaluating the manuscript. T.A.K. dedicates this article to DR. JOHN W. DRAKE, in gratitude for 15 years of insightful scientific comments, strong administrative support and sage advice as a mentor, and as a friend.

	LITERATURE CITED

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