Carcinogenesis vol.20 no.2 pp.215–220, 1999
MSH3 deficiency is not sufficient for a mutator phenotype in Chinese hamster ovary cells
John M.Hinz and Mark Meuth1
Recent studies have revealed multiple homologs of the Escherichia coli mismatch repair gene MutS in both lower and higher eukaryotes (1,2). Six MutS homologs have been identified in Saccharomyces cerevisiae (2–6). These proteins play roles in the recognition and correction of DNA replication errors or chemical damage to DNA, processing of recombination intermediates and suppression of recombination intermediates between divergent DNA sequences. Three of these homologs (MSH2, MSH3 and MSH6) are essential for the repair of DNA replication errors (2,6,7). In yeast these homologs appear to specialize in the types of damage they recognize for repair; however, there is redundancy in the recognition of some types of replication errors. A heterodimeric complex between MSH2 and MSH6 (called MutSα) appears to recognize single base pair mismatches whereas either MutSα or a complex between MSH2 and MSH3 (called MutSβ) recognizes substrates formed by single nucleotide loops. The MutSβ heterodimer binds to larger loops such as those produced by misreplication of di-, tri- or tetranucleotide arrays. Because of the central role played by MSH2 in mismatch repair, yeast cells with knockouts of MSH2 have substantially higher mutation rates than cells with
knockouts of either MSH6 or MSH3 alone (2,6). Knockouts of both MSH6 and MSH3 result in mutation rates nearly equal to those in the MSH2 deficient cells. Evidence supporting similar roles for the multiple MutS homologs found in mammalian cells has been reported (8,9); however, some of these data suggest that the human mismatch binding complexes may not have the same strict substrate specificities as the yeast complexes (10). Human cell lines obtained from tumors of hereditary non-polyposis colon cancer (HNPCC) patients, or from sporadic tumors, have been identified that are deficient in hMSH2, hMSH6 or both hMSH6 and hMSH3 (10,11). The hMSH6-deficient colorectal carcinoma cell line DLD-1 has a low level of microsatellite instability at di-, tri- or tetranucleotide repeats relative to hMSH2-deficient lines (12–14), although instability at mononucleotide runs and base substitution rates were substantially elevated (14). The elevated rate of mutation and the repair deficiency in this line could largely be corrected by the introduction of chromosome 2 (which carries a wild-type copy of hMSH6) into these cells (15). Furthermore, the defect in the repair of single base mismatches found in cell-free extracts prepared from DLD-1 was corrected by addition of the purified hMutSα complex (8). More recently, the endometrial tumor cell line HHUA, defective in both hMSH6 and hMSH3, was found to have instability at all types of microsatellites and a high rate of base substitution (10). This instability again could be substantially corrected (with the exception of instability at mononucleotide repeats) in cells and cell-free extracts by the introduction of either human chromosome 2 or 5 [which carries a wild-type copy of hMSH3 (10)]. These data suggest similar or redundant roles for hMSH3 and hMSH6, though only mutations in hMSH6 have been found (albeit infrequently) in families predisposed to colon cancer (16). In the work reported here, we specifically examined the role of MSH3 in maintenance of genomic stability by using CHO cell strains with deletions of both copies of this gene. We show that loss of MSH3 has only a limited effect on microsatellite instability. Instability was found in only one of two MSH3deficient strains and at only one locus. Furthermore, there was no consistent increase in spontaneous mutation rate at two selectable loci relative to MSH3-proficient strains. In addition, we examined the sensitivity of the deficient cells to a wide range of damaging agents to determine whether MSH3 played a role in the response to these agents. Except for a small level of resistance to 6-thioguanine (6-tg), sensitivity to other agents was not significantly affected. The negligible affect of MSH3 deficiency on microsatellite stability and mutation rate determined in this study supports the hypothesis that there is a high degree of redundancy in the repair of DNA replication errors at microsatellite repeats (10).
Abbreviations: CHO, Chinese hamster ovary; HNPCC, hereditary nonpolyposis colon cancer; IR, ionizing radiation; MEM, minimum essential medium; RT–PCR, reverse transcriptase–polymerase chain reaction; SDS, sodium dodecyl sulfate; UV, ultraviolet.
Cell culture The Pro– CHO cell line used in these experiments is the ‘Toronto strain’ obtained from Lou Siminovitch (University of Toronto). Cell lines UA41,
Departments of Oncological Sciences and Radiation Oncology, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA 1To
whom correspondence should be addressed Email:
[email protected]
In the yeast Saccharomyces cerevisiae, the mutS homolog protein products MSH3 and MSH6, each in cooperation with MSH2, play well-defined and specific roles in the repair of DNA mismatches and nucleotide loops. The discrete functions of the human homologs hMSH3 and hMSH6 are less clear and current evidence suggests that the substrate specificity of these proteins may be less strict. To determine the role of MSH3 in mammalian mismatch repair, we employed MSH3-deficient Chinese hamster ovary (CHO) cell lines. No significant changes in mutation rate were detected in the MSH3-deficient strain and there were no differences in sensitivity to DNA-damaging agents. Further analysis of hprt mutants did not show a MSH3dependent shift in the mutant spectrum. Interestingly, thorough examination of four dinucleotide microsatellite regions revealed instability at only one locus in one of the MSH3-deficient cell lines. These data support the idea of a high degree of redundancy in the function of the MutS homologs MSH3 and MSH6, at least with respect to the control of microsatellite instability. Introduction
© Oxford University Press
Materials and methods
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J.M.Hinz and M.Meuth DG44 and D35 were acquired from Larry Chasin (Columbia University) (17,18). The lines were maintained in α-minimum essential medium (MEM) (Gibco BRL, Grand Island, NY) with 10% dialyzed fetal bovine serum (HyClone) and 10 µM thymidine. DG44 was further supplemented with 20 µM hypoxanthine. Determination of MSH3 expression The presence of the full-length MSH3 transcript was determined by reverse transcriptase–polymerase chain reaction (RT–PCR) amplification of two separate regions of msh3. CHO msh3 primers were selected from regions of sequence identical in both human (HSDUG; EMBL accession no. J04810) and mouse (MMREP3B; EMBL accession no. M80360) mRNA. The amplified 59 region of the CHO transcript was located between human base 910 (784 in mouse) and 2314 (2179 in mouse). The primers for this region were 59GGTGGGAGTTGTGAAGCAAA-39 and 59-ACAGCTTTTGTGCTTCCAAC-39. The 39 region of transcript amplified for detection was located between human base 2124 (1989 in mouse) and 3078 (2940 in mouse). The primers for this region were 59-TGACTTCCCTTTAATAAAAAAGA-39 and 59-ATCCCATGTGGTAATTCCCC-39. Amplification products were separated by agarose gel electrophoresis and detected by ethidium bromide staining. Microsatellite analysis Subclones were picked from dilute plating of cell stocks. Clones were expanded and lysed with DNA lysis buffer [100 mM Tris–HCl, pH 8.3, 5 mM EDTA, 0.2% sodium dodecyl sulfate (SDS), 200 mM NaCl] and proteinase K (Gibco BRL). DNA was isolated by isopropanol precipitation and resuspended in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). PCR was used to amplify the microsatellite loci of each clone. Primers used for amplification of CHO microsatellite loci 10.1, 11.1 and 21.1 have been published (19). These products were sequenced by automated sequencing to determine length of nucleotide run. Primers used for GT23 amplification were 59-ATCTGAAGTTAAAATGAAGTTG-39 and 59-CTCTGTGGGTATGCACATAG-39 as determined from the sequence published by Nalbantoglu et al. (20). Primers were end-labeled, employing T4 polynucleotide kinase in the presence of [γ-32P]ATP. Amplified microsatellite DNA from clones were separated by polyacrylamide gel electrophoresis and detected by autoradiography. Mutation rate determination Mutation rate was determined by Luria–Delbru¨ck fluctuation analysis (21). Replica cultures were seeded with 1000 cells and grown to near confluency. The total number of cells per replica was determined by counting on a hemacytometer and all cells were plated in selective medium. Approximately 13106 cells/100 mm plate were plated in 2 mM ouabain. Approximately 53105 cells/100 mm plate were plated on 1 µg/ml 6-thioguanine (6-tg). Colonies in each replica were counted after 7–10 days of growth. Mutation rate was calculated by both Po (21) and ‘method of the mean’ calculations (22). Dose–response Cells (100–1000) were plated and exposed to increasing concentrations of selective agent. cis-platinum and 6-tg exposure was continuous throughout the experiment. Cells were exposed to MNNG for 1 h in serum-free medium 24 h after plating. The surviving fraction was calculated using number of colonies, growing after 7 days, at a given concentration of selective agent divided by the number of colonies growing in non-selective medium. To determine ultraviolet (UV) radiation resistance, the medium was removed from 100 or 1000 cells, which were plated 24 h earlier, and exposed to a UV source (254 nm, 1 mJ/cm2/s) for increasing amounts of time. All cells were without medium for equal amounts of time, with variation only in UV exposure time, with doses ranging from 0 to 2.0 mJ/cm2. Ionizing radiation (IR) resistance was determined by plating 100 or 1000 cells after exposure to varying doses of IR. Tubes of cells suspended in medium were exposed for varying amounts of time to γ-radiation (137Cs, 5.75 Gy/min) at doses ranging from 0 to 12 Gy. hprt mutant analysis RNA was isolated from 6-tg resistant subclones of D35, amplified by RT– PCR and sequenced to determine the precise hprt mutations. Sequence was determined by automated sequencing using primers for internal and external overlapping sequences. The primers used for sequencing were as follows: 59 external forward primer, 59-TTCCTCCTCACACCGCTCTT-39; 39 external reverse primer, 59-TGCAGATTCAACTTGAACTCTC-39; 59 internal forward primer (starting at base 339), 59-AGCACTGAATAGAAATAGTG-39; and 39 internal reverse primer (starting at base 549), 59-GTACCGCTTGACCAGGGAAAG-39. Mutations were determined by alignment to the wild-type sequence with the Sequencher program (GCC). Cell growth in HAT medium (50 µM hypoxanthine, 1 µM aminopterin, 10 µM thymidine) was determined for 6-tg resistant D35 clones that had no detectable hprt mutations.
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Fig. 1. RT–PCR analysis of the MSH3 transcript in Pro–, UA41 and D35. Two distinct regions (59 and 39) of MSH3 were amplified for detection in each cell line.
Results Cell lines The CHO cell strains deficient in MSH3 used in this work were isolated by Chasin et al. through the selection of deletions that eliminated the adjoining and divergently transcribed dihydrofolate reductase (dhfr) gene (23). This was accomplished through a multi-step procedure beginning with the CHO strain Pro– (17). A mutant strain of Pro– containing amplified dhfr was used to select a strain (UA41) sensitive to the inhibitor methotrexate, as a result of the deletion of amplified dhfr array and surrounding sequences. The radiation-induced mutant strain (DG44) deficient in the remaining dhfr allele was produced by a second selection for methotrexate-sensitive cells (18). A deletion of at least 115 kb was identified in DG44, which encompassed most (if not all) of dhfr, and a 10 kb sequence upstream of dhfr, which encodes MSH3. As a result of the loss of dhfr, DG44 requires an external source of purines and thymidine for growth. Since inclusion of purines in the growth medium precludes measurement of hprt mutation rate by 6-tg selection, a derivative of DG44 (called D35) that contains a dhfr expression construct (24) was used. Msh3 transcripts were not detectable in this cell line by RT–PCR analysis (Figure 1). We compared the properties of msh3 deficient (msh30/0) D35 and DG44 cells with the parental CHO strains Pro– (which has two copies of msh31/1) and UA41 (which has a single copy, msh30/1). Microsatellite instability Dinucleotide repeats at four CHO loci were examined in msh3 proficient and deficient strains. DNA purified from subclones was used to amplify these repeats in order to determine the frequency of cells with novel alleles. The frequency of novel alleles in subclones isolated from the Pro– parental or UA41 was low or undetectable (Table I). Novel alleles were significantly more frequent (P , 0.001) in subclones isolated from DG44 relative to the three other strains (Pro–, UA41 or D35; Table I). However, one locus (21.1) accounted for most of the novel alleles found in DG44 (Figure 2). Elimination of this locus from statistical analysis showed the level of instability in DG44 to be significantly greater than only that of UA41 (P , 0.025). Since there are no significant differences (P . 0.05) in the frequency of novel alleles at the four loci among the other cell strains, 21.1 appears to be a mutation hotspot in
MSH3 deficiency and a mutator phenotype
Fig. 2. Analysis at microsatellites in subclones of DG44. Locus 10.1 (A) shows no novel alleles, locus GT23 (B) shows three mutant alleles and locus 21.1 (C) shows 18.
Table I. Microsatellite analysis in MSH3-proficient and -deficient cells at four different (CA)n microsatellite loci Locus
10.1 (n 5 25) 11.1 (n 5 22) 21.1 (n 5 20) GT23 (n 5 23) Total (%)
Cell line (MSH3 status) Pro– (1/1)
UA41 (0/1)
D35 (0/0)
DG44 (0/0)
Total
0/68 1/68 2/68 0/68 3/272 (1.1%)
0/74 0/74 0/74 0/74 0/296 (,0.03%)
1/69 3/69 2/69 2/69 8/276 (2.8%)
1/51 0/51 25/51 4/51 30/204 (14.7%)
2/262 4/262 29/262 6/262
Mutant clones are represented as a fraction of total clones tested. Primers for these hamster-specific microsatellite loci have been published previously (19). The number (n) of CA repeats in loci 10.1, 11.1 and 21.1 were determined by sequencing the region in both Pro– and D35 cell lines. The GT23 sequence was published previously (20). Between the cell lines, DG44 is significantly different from all other cell lines (P , 0.001) and UA41 is significantly different from D35 (P , 0.01). Disregarding the highly unstable 21.1 locus, UA41 is still significantly different from both D35 (P , 0.05) and DG44 (P , 0.025), but no other loci vary significantly. Between the loci, 21.1 is significantly different (P , 0.001) from all other loci. Disregarding the cell line DG44, in which instability was high at the 21.1 locus, there is no significant difference between any loci.
DG44. D35 had a significantly increased frequency of novel alleles compared with UA41, but not Pro–. Since the increase in frequency of novel alleles at 21.1 occurred in DG44, which was grown in the presence of 20 µM hypoxanthine and 10 µM thymidine, we considered the possibility that ribo- and deoxyribonucleotide triphosphate pool alteration was responsible for this effect. To test this, Pro– and D35 (which did not show this hotspot) were grown in varying concentrations of hypoxanthine (20–100 µM) and thymidine (20–100 µM). DNAs from the cells grown in these conditions were collected and the hotspot locus, 21.1, and a
locus showing a low level of instability, 10.1, were assayed for novel alleles. No instability was detected at either locus. Interestingly, novel alleles of the four microsatellite loci were also not detected in 20 clones obtained from each of the CHO mutator strains previously shown to have increased mutation rates [methotrexate-resistant Pro– MTXr (25,26) and thymidine-dependent mutator strain Thy–49 (27)]. Thymidine is known to directly affect mutation rate at DNA coding sequences in Thy– 49 and the concentrations used in the isolation of these subclones give maximal effects (27). Mutation rates at selectable loci To determine whether MSH3 deficiency affected mutation rates at coding sequences, we examined the rate of mutation in these strains to 6-tg and ouabain resistance by the Luria– Delbru¨ck fluctuation test. In these experiments, replica cultures inoculated with 1000 cells each (to eliminate pre-existing mutants) were grown to high cell densities. All cells from each replica culture were plated in one of the selective agents. Mutation rate was determined using Po and method of the mean calculations (see Materials and methods). As shown in Table II, mutation rate was slightly higher at the ouabain resistance (OuaR) locus in the MSH3-deficient DG44 but not in D35. The hprt mutation rate was not determinable in DG44 due to its purine requirement; however, the hprt mutation rate in D35 was not significantly different from the wild-type. This suggests that the increase in mutation rate in DG44, at the OuaR locus, is not a result of MSH3 deficiency alone. The spectra of hprt mutations in D35 The spectra of spontaneous hprt mutants in D35 was also determined. Although the mutation rate was not significantly increased, in this strain there may have been a subtle change in the nature of the mutations. For example, as MSH3 is known to play a role in prevention of recombination between non-identical DNA sequences in yeast (7), as well as runs of 217
J.M.Hinz and M.Meuth
Table II. Calculated mutation rates of CHO cell lines at hprt and OuaR loci as determined by fluctuation analysis in the presence of 6-tg or ouabain, respectively Cell line
Pro–
UA41
D35
DG44
(MSH3 status) Plating efficiency (%) Doubling time (h) Locus Mean no. cells/replica Replicas Replicas with mutants Mutant frequency Mutation rateb (STD) Mutation ratec (STD)
(1/1) 51 12.0 hprt 2.73106 52 30 1.0310–5 1.6310–7 (6310–8) 5310–7 (4310–7)
(1/0) 56 10.7 hprt 1.03106 48 22 6.7310–7 4.2310–7 (1310–8) 1.6310–7 (3310–8)
(0/0) 49 13.5 hprt 1.33106 39 14 1.5310–5 2.5310–7 (4310–8) 1.5310–6 (6310–7)
(0/0) 29 18.0 hprt
OuaR 2.43106 13 2 ,1.3310–7 3.5310–8 (ND) 6.7310–8 (ND)
OuaR 2.83106 16 1 ,1.3310–7 1.6310–8 (ND) 5.4310–8 (ND)
OuaR 3.83106 21 7 1.3310–7 7.4310–8 (ND) 9.0310–8 (ND)
NDa NDa NDa
OuaR 1.33106 47 26 ,1.0310–7 4310–7 (2310–7) 4310–7 (1310–7)
aPurine
requirements of DG44 preclude hprt mutant analysis. calculated by Po. calculated by method of the mean. ND, not determined; STD, standard deviation of the trials.
bAs cAs
Table III. Mutations at the hprt locus in 6-tg resistant D35 clones Clone Transitions MTG21 MTG13 Transversions MTG25 DHTG4 DHTG3 MTG6 DHTG 18 DHTG14 Frameshifts DGTG12
Mutation nta (exon)
Context
Alteration
A→G G→A
80 (ex 2) 568 (ex 8)
AATC A CTAT TGTT G GATA
His→Arg Gly→Arg
T→A T→A C→A G→T A→T T→G
62 305 577 580 598 615
GATT T ATTT AGAC T GAAG TGCC C TTGA CCTT G ACTA CTTC A GGGA ATAT T TGTG
Leu→stop silent Leu→Tyr Asp→Tyr Arg→stop Ile→Met
GACT gtaac acag TTGTd
Terminates in four codons
ND ND TAT AAT GAG
Insert ex 2 & 3 Ex 7 loss ∆ Asn
1G
(ex (ex (ex (ex (ex (ex
2) 3) 8) 8) 8) 9)
532b (ex 7 or 8) Duplications and deletions DHTG1 ND ND DHTG9 ND ND TG1 ∆ AAT 586 (ex 8)
ND, not determined. aNucleotide position based on cDNA sequence (EMBL accession no. J00060). bPosition in cDNA at exon 7/8 border, genomic positioning not determined. cSpace represents possible insertion site at 39 end of exon 7. dSpace represents possible insertion at 59 end of exon 8. Genomic sequences are from ref. 45.
simple sequence repeats (7), there may be an increase in intragenic exchanges within hprt that leads to loss of coding sequences. To test this possibility we isolated independent 6tg-resistant D35 mutants obtained from the replica cultures described above. RNA purified from these mutants was then used in RT–PCR reactions to obtain cDNAs. Of 25 mutants that gave cDNA products, mutations were found in 12. The frequency of cDNAs without detectable hprt mutations was unusually high. In our past studies, mutations were not detected in only ~10% of cDNAs (28). To test the possibility that these strains became resistant to 6-tg by a mechanism that did not involve hprt [such as a mismatch repair deficiency, which can confer a low level of 6-tg resistance (29)], we tested the ability of these mutants to grow on HAT medium. Of seven tested, two did not grow on HAT, which is consistent with these strains being deficient in hprt. The other five were not sensitive, 218
which demonstrates that they were not hprt deficient. These strains may be the result of mutations or even epigenetic alterations in the promoter region of hprt that downregulated its expression. Of the remaining five that grew in HAT, the three tested were sensitive to 6-tg, which demonstrates that they were not stably 6-tg resistant. Of the cDNAs with detectable sequence alterations at hprt, base substitutions resulting from both transitions and transversions were found, although transversions were the dominant mutation type (50%) in our small sample (Table III). A frameshift resulting from a 1G insertion was found, but this did not occur in a run of G residues. One mutant gene contained a 3 bp deletion, but not in a region of repeats. Another mutant lost exon 7, although the precise mutation resulting in exon loss was not determined. A duplication of exons 2 and 3 was detected in the last mutant characterized. This particular duplication has been reported before (30), and like other gene duplication mutations in hprt (31), this strain had a high reversion frequency (2310-5). Thus, compared with mutations previously reported for wild-type CHO cells, there was no striking shift in the nature of the mutations accumulating among this small collection of mutant strains. Importantly, the MSH3-deficient lines did not develop frameshift hotspots like those found in mismatch repair-deficient human tumor cell lines (10,13,28,32). Sensitivity to DNA damaging agents In recent reports, tumor cell lines deficient in various components of the mismatch repair pathway have been shown to have altered sensitivity to a wide range of DNA-damaging agents. It has been proposed that these alterations may result from participation of the MMR proteins in the recognition and (attempted) repair of base damage induced by alkylating agents or base analogs (29,33,34), transcription coupled nucleotide excision repair (35), or cell cycle checkpoint controls (29). As an initial approach to determine whether MSH3 played a similar role, we examined the sensitivity of Pro– (MSH31/1), UA41 (MSH30/1) and D35 (MSH30/0) to mutagenic agents. Figure 3 shows that D35 cells were slightly more resistant (1.7-fold, D10 0.071 µg/ml) to 6-tg but not MNNG. UA41 cells were as sensitive as wild-type Pro– to 6-tg (with D10 values of 0.042 and 0.046 µg/ml, respectively). Sensitivities to other agents (UV light, IR or cis-platinum) were not significantly altered (data not shown).
MSH3 deficiency and a mutator phenotype
Fig. 3. Effect of 6-tg and MNNG on survival of MSH3-proficient and -deficient strains. (A) Cell survival in response to 6-tg. (B) Cell survival in response to MNNG. Pro– is represented by u; UA41 is represented by d; D35 is represented by m.
Discussion Human tumor and mouse cell lines deficient in the mismatch repair proteins MLH1 or PMS2 develop mutator phenotypes characterized by a significantly increased rate of mutation at microsatellite and coding sequences (13,36,37). The effects of loss of the MutS homologs is more complicated. Embryonic fibroblast lines developed from mice deficient in Msh2 have an increase in the frequency or rate of mutation (38,39); however, human tumor cell lines deficient in this repair protein do not show a consistent change in mutation rates (37,40). In some of these human tumor cells the mutation rate has been shown to be dependent on certain environmental conditions (40). In the human endometrial tumor cell line HHUA, the increased mutation rate appears to depend on loss of both hMSH6 and hMSH3, as the mutator phenotype can be largely corrected by complementation with the human chromosome bearing either gene (10). In contrast, DLD-1, deficient in hMSH6 but proficient in hMSH3, has at least a 100-fold increased mutation rate (13,37). Depletion of the hMSH2/
hMSH6 heterodimer by overexpression of hMSH3 increased the hprt mutation rate as much as 1000-fold (41). The work presented here supports the idea of a high degree of redundancy in the function of the mismatch repair proteins MSH3 and MSH6, at least with respect to the control of microsatellite instability. One of the hamster cell strains lacking MSH3 (DG44) had a 10-fold increase in mutation rate at the OuaR locus; however, D35, which also lacks MSH3 but has had DHFR restored, showed little or no change in mutation rate. Thus the increase in mutation rate does not appear to be the result of MSH3 deficiency. This is consistent with the preference of the MSH2–MSH6 complex for the recognition of the DNA replication errors that give rise to the mutations in the OuaR locus. Also, little or no change was observed in dinucleotide repeat instability that can be attributed to MSH3, in contrast to the observations in yeast, where loss of MSH3 increases the rate of mutation in simple sequence repeat arrays (7). It is also consistent with the observation that the MutSα complex is able to bind and presumably repair some types of loops (8). Instability was detected in one cell strain deficient in MSH3 (DG44) but not another (D35). Thus the instability detected at the single locus in DG44 may be the result of some other alteration. Since this DG44 is deficient in dhfr, the instability in the microsatellites as well as at the OuaR locus may even be the result of some sort of ribo- or deoxyribonucleotide pool imbalance resulting from the loss of this enzyme and the supplementation of the medium with hypoxanthine and thymidine. However, changes in the thymidine and hypoxanthine levels in D35 and Pro– did not enhance microsatellite instability. Furthermore, no instability was detected at any of the four loci tested in a CHO strain (Thy-49) known to have an increase in mutation rate as a result of pool alterations (27). Mismatch repair-deficient lines derived from human tumors or mice display altered sensitivity to a wide range of DNAdamaging agents and base analogs (29,33–35,42,43). Our results indicate that MSH3 is not involved in these altered responses because the MSH3 deficient hamster cells showed little or no change in sensitivity to these agents. This is not unexpected given that lesions induced by MNNG or 6-tg are specifically recognized by the MutSα complex (MSH2–MSH6) (44). There was slight resistance to 6-tg by D-35, but again we cannot eliminate the possibility that this might be the result of altered purine pools. Inherited mutations of hMSH3 have not been found in HNPCC patients. Mutations in this gene have been detected in sporadic tumors, but only in association with mutations of other mismatch repair genes (10). Our studies are consistent with the idea that limited effects of MSH3 deficiency may be the result of the high degree of redundancy in the functions of the mutS homologs in mammalian cells. Acknowledgement This work was supported by grants (CA22188 and CA62244) from the National Cancer Institute to M.M.
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