A Mutation in the MSH5 Gene Results in Alkylation Tolerance1

[CANCERRESEARCH57,2715â€”2720,July1. l997J A Mutation in the MSH5 Gene Results in Alkylation Tolerance1

Sonya Bawa and Wei Xiao2

Department of Microbiology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada

ABSTRACT results in the accumulation of single-stranded nicks in DNA, which are ultimately lethal to the cell. This hypothesis is attractive not only DNA methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine because it provides an alternative to 06 MeG/O4 MeT genotoxicity (MNNG)are potent carcinogens;their carcinogeniceffectis mainlydue to but also because genetic defects in several human MMR genes have the effect of production of O'-methylguanine (06 MeG) on DNA. 06 MeG is not only mutagenic but also toxic to the cell because Mer/Mex cells been linked to hereditary nonpolyposis colon cancer as well as other unable to remove O@MeG are very sensitive to killing by MNNG. It has types of cancers (9â€”14).However, the abortive MMR hypothesis been proposed that repeated futile mismatch correction of 06 MeG should be viewed with caution because the current supporting cvi containing bp is responsible for the genotoxicity of the O@MeG lesion and dence with mammalian cells is not conclusive and a convenient that loss of mismatch repair activity results in cellular tolerance to 06 mammalian system is not available to vigorously test the hypothesis. MeG, but the hypothesis has not been proved. We used yeast as a model The MMR system consisting of Escherichia coli MutS and MutL to test this hypothesis and found that chromosome deletion of any known homologues has been extensively studied recently in yeast and human nuclear mitotic mismatch repair genes, including MUll, MSH2, MSH3, cells (reviewed in Refs. 14 and 15). Saccharomyces cerevisiae con MSH6, and PMSJ, did not rescue mgtlA 06 MeG DNA repair methyl tains six MutS homologues (Msh) and three MutL homologues. Mshl transferase-deficient cells from killing by MNNG. A large number of is responsible for mitochondrial DNA repair (16). Msh2, Mlhl, and mgtTh, MNNG-tolerant revertants were isolated, among which one cell line, XS-14, has been found to carry a mutated allele of the MSH5 gene. Pmsl are required for the correction of various mismatches because The mutation also affected spore survival but did not increase the spon mutation in any of the three genes results in a markedly enhanced taneous mutation rate. We further demonstrated that a mutated form of spontaneous mutation rate or instability of simple repeats, which is not the MSHS gene, mshsâ€”14, not the msh5@-null mutation, is responsible for further increased in the double mutants (16â€”18).In contrast, Msh3 the cellular tolerance to MNNG in XS-14 cells. This observation offers an and Msh6 appear to form alternative pathways to correct 2â€”4-bp alternative model that may reconcile seemingly contradictory observa insertions and deletions or l-bp mismatches, respectively (19, 20). lions of yeast and mammalian cells. Msh4 (21) and MshS (22) have been shown to facilitate meiotic reciprocal recombination but are believed to play no role in the actual INTRODUCTION process of mismatch correction. The abortive MMR hypothesis has been tested in yeast, which is The genome of the cell is dynamic and reacts readily with physical relevant to mammalian systems in that all MTase and MMR proteins and chemical agents, resulting in modifications to its molecular struc are highly conserved between the two. It was found that none of the ture. DNA alkylating agents are present as one of the largest classes msh2iX, msh3& mlhM, or pms1@-null mutations could rescue mgt1@s of environmental chemical carcinogens; some of the alkylating agents strains from MNNG-induced killing and that MMR defects were not are also produced endogenously as a consequence of cellular meta required for the selection of MNNG-tolerant clones (23). The conflict bolic processes. Challenging cells with S@l-type methylating agents between the observations of yeast and human cells suggests that either such as MNNG3 and N-methyl-N-nitrosourea produces mutagenic and the abortive MMR hypothesis is not universal or 06 MeG genotox carcinogenic lesions like @6MeG and (to a lesser extent) O@ MeT, icity is the result of recognition/repair of 06 MeG by a mechanism which, if not corrected by the DNA repair MTases, pair with thymine other than MTase that interferes with DNA replication and results in and guanine, respectively, resulting in transition mutagenesis (1, 2) cell death. The former possibility seems unlikely, because in the and carcinogenesis in mammals (3). absence of MTase the lesion is equally toxic in all organisms studied In the absence of Mlase, bacterial, yeast, and mammalian cells to date (3â€”5,24). It also remains possible that 06 MeG toxicity is due become extremely sensitive not only to mutagenesis but also to killing to the involvement of yet to be identified MutHLS homologues or to by MNNG, which cannot be explained simply on the basis of induced the specific mutations of known MutHLS homologous genes. mutation rates (3â€”5).Itis thus evident that persistence of @6MeGand We report here an attempt to further investigate the mechanism of possibly O@MeT lesions is genotoxic, although the mechanism of cell alkylation toxicity and its relationship to MMR. We have isolated and death is not yet clear. The MMR system has been implicated in the characterized a large number of MTase-deficient, MNNG-tolerant cell processing of 06 MeG-containing bp into lethal lesions, because lines; one of the extensively studied mutants appears to carry a recent studies with MerTh'Mex mammalian cells lacking MTase mutation in the MSH5 gene that is associated with the MNNG resist activity (6, 7) suggest that alkylation tolerance is accompanied by loss ant phenotype. of normal MMR activities. This correlation was predicted by the abortive MMR hypothesis (8), which suggested that repeated futile attempts to correct mispairs during replication of a damaged template MATERIALS AND METHODS

Received 11/18/96; accepted 4/29/97. Plasmids and Yeast Transformation. Plasmid pWX1149 carries the The costs of publication of this article were defrayed in part by the payment of page MGTJ gene in a single-copy YCp vector. YCp-MLH1 (17) was from Dr. M. charges. This article must therefore be hereby marked advertisement in accordance with Liskay (Oregon Health Sciences University, Portland, OR), p11-2 (YCp 18 U.S.C. Section 1734 solely to indicate this fact. MSH2; Ref. 25) was obtained from Dr. R. Kolodner (Harvard Medical School, I This research was supported by National Cancer Institute of Canada Grant NC1C007412(to W. X.). W. X. is a Research Scientist of the National Cancer Institute of Cambridge, MA), pWBK3-PMSl(YCp-PMS 1; Ref. 26) from Dr. W. Kramer Canada. (Georg-August University, Gottingen, Germany), YCp-MSH3 (27) and YCp 2 To whom requests for reprints should be addressed. Phone: (306) 966-4308; Fax: MSH6 from Dr. 0. Crouse (Emory University, Atlanta, GA) and YCp-MSH4 (306) 966-4311; E-mail: [email protected]. (21) was from Dr. S. Roeder (Yale University, New Haven, CT). Plasmid 3 The abbreviations used are: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; 0Â° MeG, 0Â°-methylguanine;0' MeT, 0â€•-methylthymine;MMR,mismatch repair; MTase, pNHl89â€”2 (22) was a gift from Dr. N. Hollingsworth (State University of 0Â°MeG/04 MeT DNA repair methyltransferase; Msh, MutS homologue. New York, Stony Brook, NY). A 4.5-kb BamHI/HindIII fragment containing 2715

strainsStrainGenotypeSourceWXY9IO2MATa Table I S. cerevisiae

54XS-803-2CMATa ura3-52 trpl-M3 leu2-@I mgtM ::LEU2 GAL@Ref. GietzaXS-803-3AMATa leu2-3.112 ura3-52 hisl-2 hom3-lO canirD. Gietz2CrngtI@sLike leu2-3,112 ura3-52 his/-I trp2 canisD. study3AnzgtlL@&Like XS-803-2C but with mgtM::LEU2This study2Crngtl@unlhLXLike XS-803-3A but with mgtM ::LEU2This study2Cmsh5Like XS-803-2C but with mgtM::LEU2 mlhM:: URA3This study2C1ngtl@?nsh5Like XS-803-2C but with msh5 :: URA3This study3Amsh5Like XS-803-2C but with mgtliX::LEU2 msh5::URA3This study3Aingth@sinsh5Like XS-803-3A but with msh5::URA3This study2Cmsh6@Like XS-803-3A but with mgt1@::LEU2 msh5:: URA3This study2C,ngtI@sinsh6@iLike XS-803-2C but with ,nsh6/s ::hisG-URA3-hisGThis study2CmgzI@ansh5@sLike XS-803-2C but with mgtlts ::LEU2 mshÃ³ts::hisG-URA3-hisGThis studyXS-l4MNNG-resistant XS-803-3A but with mgtI@::LEU2 msh5@::hisGThis mutant of 2CmgtIiX ::LEU2This study â€œUniversityof Manitoba. Canada.

the entire MSH5 gene was cloned into plasmids YCplac33 and YIplac2l 1 (28) onine to score Hom@ revertants from hom3â€”1Omutants and on YPD for total to form YCp-MSH5 and YIp-MSH5, respectively. Yeast transformation was as cell survival. described previously (23). Plasmid pmgtl@::LEU2 (5) was used to create Quantitation of Cellular Glutathione. Crude yeast cell extract was pre mglU@ strains. pmsh5@::hisG-URA3-hisG was constructed by removing a pared as described previously (32). Glutathione content was estimated by the 0.84-kb CIaI-EcoRV fragment from the MSH5 coding region and inserting a method of Hissin and Hinf (33). 3.8-kb BamHI-Bg(II hisG-URA3-hisG fragment from pNKY5 I (29) at the unique Bg!1l site. Strains and Cell Culture. The S. cerevisiae strains used in this study are RESULTS listed in Table 1. All mgtI@::LEU2 mutants were created as described (5). The The msh6@ Mutant Does Not Rescue mgtTh from Kiffing by msh6@smutants were obtained by SphI+EcoRI digestion of plasmid MNNG. Our previous results had shown that deletion of any of the pmsh6@::hisG-URA3-hisG, constructed by Dr. W. Kramer.4 Two msh5 dis ruption alleles were created. The MSH5 gene was interrupted in msh5::URA3 then known MMR genes (i.e., PMSJ, MLHJ, MSH2, and MSH3) at the unique Bg!lI site using pNH19O as described (22). The msh5iX mutants could not rescue the mgt1t@mutant from MNNG-induced killing. The were obtained by HpaI+EcoRI digestion of pmsh5i@::hisG-URA3-hisG. Cells MSH6 gene has since been isolated from both humans (34, 35) and containing the msh5@s::hisG-URA3-hisG allele were cultured on 5-fluoro yeast (19, 20). This gene is particularly interesting because hMsh6 orotic acid plates (30) to select for ura3 deletions (msh5@::hisG). All of the was characterized as a G:T mismatch binding protein, GTBP (34), and targeted gene disruption mutants were created by enzyme digestion of the a similar activity has been shown to recognize 06 MeG and O@ plasmid carrying the disruption cassette prior to yeast transformation, and the MeT-containing mispairs (36). Furthermore, a mutation in each genomic structure of the deletion mutants was confirmed by Southern hybrid hMSH6 allele was reported in the MNNG-tolerant cell line MT1 (37), ization. Culture media used in this study were YPD (1% yeast extract, 2% suggesting that Msh6 may be involved in 06 MeG toxicity. To test Bacto-peptone, 2% glucose) and synthetic SD (0.67% yeast extract without this hypothesis in yeast, MSH6 was deleted in a MTase-deficient nitrogen bases, 2% glucose) supplemented with amino acids and bases as background. We observed that the msh6i@smutant behaves like other recommended (3 1). To make plates, 2% Bacto-agar was added. All incubations were carried out at 30Â°C. MMR mutants in that the msh6L@ssinglemutant did not confer MNNG Mutant Isolation and Cell Killing. MNNG was purchased from Sigma sensitivity, and loss of Msh6 function did not result in tolerance of the Chemical Co. (St. Louis, MO). Stock solution of 1 mg/ml was made in 100 mt@i mgtM cells to MNNG (data not shown). acetate buffer, pH 5.0, aliquoted, frozen, and thawed only once. For both Isolation and Characterization of MTase-deficient, MNNG-tol mutant isolation and cell killing, the yeast cells were cultured overnight in 2 ml erant Mutants. Because none of the MMR mutants resulted in tol of YPD at 30Â°C.Fiveml of YPD were inoculated with 200 @xlofthe overnight erance to MNNG, we decided to isolate MNNG-tolerant mutants from culture and allowed to grow for 4â€”6h to reach a cell density of 2 X l0@ mgt1i@scells. Strains XS-803â€”2Cand XS-803â€”3Awere chosen for this cells/mi. For mutant isolation, the cells were treated with I xg/ml MNNG for study due to their ability to sporulate well. The 2CmgtM strain was 20 mm, washed, resuspended, and plated on SD plates containing 2 jxg/ml used for the isolation of MNNG resistant clones, 55 of which retained MNNG. Cell survival was monitored by plating an appropriate dilution of the their resistant phenotype upon repeated subculturing. Crossing each of culture before and after treatment on YPD plates. Colonies appearing on SD + MNNG plates after 3 days at 30Â°Cwerefurther analyzed. Two methods these mutants with the 3Amgt1i@ strain and comparing MNNG resist for quantitating cell killing were used. For liquid killing, freshly grown cells at ance of the diploids with control crosses (XS-803â€”2C X 3AingtU@ a concentration of 2 X l0@cells/ml were treated with 40 @xg/mlMNNGfor and 2Cmgt1@sX 3AmgtM) identified 10 recessive, 2 codominant, and various times, washed, diluted, plated on YPD, and incubated at 30Â°C.For 43 dominant mutants. We were unable to use the recessive resistant gradient plate assays, a gradient was created by pouring 30 ml of SD + MNNG strains described above for complementation analysis as planned, medium into a tilted, square Petri dish. After solidification, the plate was because although the diploids of some representative mutants with placed level and overlaid with 30 ml of SD medium. Overnight cultures were 3AingtM of opposite mating type had high level of sporulation printed across the gradient using microscope slides, and the plates were (>80%), spore survival was extremely low. incubated for 3 days. The length of growth across the gradient was measured, Because alkylation tolerance was thought to be a result of faulty and relative growth was expressed as a percentage of full-length growth on the plate without MNNG. MMR processes, some of the recessive resistant mutants were sub Spontaneous Mutagenesis Assay. Overnightculturesof XS-803â€”2Cand jected to a spontaneous mutagenesis assay. The assay, which scored its derivatives were inoculated into five tubes containing 10 ml of YPD each, reversion of hom3â€”1O auxotrophy, was performed to detect the mu to a final concentration of 20 cells/ml. The tubes were incubated at 30Â°C until tutor phenotype characteristic ofpmsl, mi/il, or msh2 MMR mutants a concentration of 2 X 108 cells/mI was reached. The cells were harvested, (16, 17, 26). As shown in Table 2, the mgrth strain shows a 4-fold washed, resuspended, and plated on SD medium lacking threonine and methi increase in spontaneous mutation rate over the wild-type level, whereas the mlhM mutant increases the mutation rate by about 4 W. Kramer, personal communication. 1000-fold. Of the four MNNG recessive resistant strains tested, only 2716

Table 2 Spontaneous mutation rates of S. cerevisiae strains grounds to determine whether reduced spore survival was due to strain All the strains are derivatives of XS-803-2C carrying the hom3-1O mutation. The background, deletion of MGTI, or the mutation in XS-14. Table 3 spontaneous mutation rate was calculated according to the method of Williamson et al. (55). The results were an average of three sets of experiments except for XS-64 and demonstrates that reduced spore survival accompanies XS- 14. Thus, XS-53, which were from one experiment. in addition to MNNO resistance, XS-l4 also acquires a defect in rateXS-803-2CWild-type0.412CmgtltsmgtM1.642CmgtMmlh1i@iStrainKey allelesMutation rate (X 10Â°)Relative meiosis. A Mutant Allele of MSH5 in XS-14, msh5â€”14,IsResponsible for MNNG Tolerance. Because mutationsin most MMRgenes in mlhM deed affect meiotic phenotype (15), we reasoned that if XS-l4 con XS-23 mgtlA, MNNGR 94.0 235 XS-64 mgtM, MNNGR 0.5 1.3 tains a mutation in one of these MMR genes, transforming XS- I4 with XS-53 mgtM, MNNGR 3.3 8.3 one of the known MMR genes will make XS-l4 sensitive to killing by XS-14mgtl/s, mgtIi@&,MNNGR430.0 2.11078 5.3 DNA alkylating agents. As shown in Fig. I, introduction of a single copy of the wild-type PMSI, MLHI, MSH2, MSH3, or MSH6 gene into XS-l4 had no effect on its resistance. To our surprise, MSH4 60 partially reduced the MNNG tolerance, but maximum functional 50@ complementation was conferred by transformation of the MSHS gene. The presence of a single copy MSHS in the XS- 14 strain significantly reduced its alkylation tolerance compared to the untransformed strain. This result was confirmed by repeated nonselective culturing of ! XS-l4/YCp-MSH5 for plasmid loss, which was accompanied by 0 30 XS-l4 regaining its resistant phenotype. We created the msh5-null mutation in ingtI@ strains and did not observe an increased MNNG resistance (Fig. 2, Lanes 2â€”4).Thus, it @ 10@ was important to determine whether the phenotype of XS-l4 was due to a mutation within the MSH5 structural gene or to a secondary 0â€¢ (suppressor) mutation. We designed the following experiments to @@@ U â€˜- ei @@@ Â°.4 ,.@ ,.:â€˜ address this question. First, it was known that the wild-type strain and the MSHS gene were able to suppress MNNG tolerance in XS-l4. If the XS-14 e@ mutation was within the MSHS gene, one would expect a lack of

Fig. I. Relative resistance of XS-803â€”2Cand its derivatives to 10 jsg/ml MNNG on a complementation by the msh5-null mutant. Indeed, the mgll@ single gradient plate. The gradient plates were incubated at 30Â°Cfor 72 h. Each experiment was mutant was able to complement the mutation in XS- 14, whereas the repeated with at least three independent transformants, and a typical result is presented. Transformants with single-copy plasmids containing different MMR genes are shown.

Table 3 Spore s'iabilitv of control and XS. /4 .roSS@S XS-23 exhibited a mutator phenotype. However, transformation of sporesXS-803-2CDiploidViable spores/total XS-23 with any of the known nuclear MMR genes (MSH2, MSH3, XS-803-3A20/202Cmgth@sx MSH6, MLHJ, and PMSI) did not significantly alter its MNNG XS-803-3A20/202CmgtI@&X resistant phenotype (data not shown). 3AmgtI@20/20XS-803-2CX WXY9IO220/202CmgtMx Phenotype of XS-14. The XS-14 recessive resistant strain was WXY9IO220/20XS-l4X selected for detailed study because it was the most MNNG resistant X3AmgtI@0/40XS-l4 X WXY9IO2/40 among the recessive mutants isolated and it did not exhibit the mutator phenotype. As a matter of fact, XS-14 was more resistant to killing by MNNG than its wild-type strain (Figs. 1 and 3). It also exhibited a 60. slow-growth phenotype with a doubling time of 3.83 h, compared to 1.99 h for the parental strain 2Cmgli& It has been shown that the t_ 50.

alkylating activity of MNNG is potentiated in the presence of thiols; â€˜C thus, reduced intracellular levels of acid-soluble thiols, such as glu @4o. 0 tathione, may also be responsible for enhancing the cellular resistance C., 30. to MNNG (38). To determine whether the resistance of XS-l4 is a 0 consequence of the above mechanism, intracellular glutathione con :; 20 tent of XS-l4 was measured and found to be comparable to that of the 0 0 @ wild-type and mgtl-deleted strain from which it was isolated (data not 10. shown). Thus, the alkylation tolerance of XS-l4 is not due to altered MNNG metabolism. Furthermore, transformation of XS-l4 with a 0. wild-type copy of the MGTJ gene enhanced its resistance to MNNG, L I indicating that the mutated gene in XS-l4 and MGTJ belong to Strain

different epistatic groups. Hence the XS-14 mutation confers resist Fig. 2. Relative resistance of different haploid and diploid strains to 10 @ig/ml MNNG ance by a pathway distinct from MTase. on a gradient plate. The gradient plates were incubated at 30Â°Cfor 72 h. Strain I, The XS-l4 mutant was crossed with the 3A,ngt1@ strain of oppo XS-803â€”2C; strain 2, 2CmgtM; strain 3) 2@mgtJdansh5@; strain 4, 3AmgzI@,nsI:5; strain 5, 2CmgtM X 3AmgtI& strain 6, 2CmgtI@ X 3Amgt1@nash5; strain 7, site mating type to conduct genetic analysis. The XS-l4/3Amgi'M 2Cmg:[email protected] x 3A,ngiIi@smsh5; strain 8. XS-l4 X 3Anagt1@; strain 9, XS diploid sporulated well, forming a high percentage of four-spore 14 X 3AmgtMmsh5. Each strain was tested with three individual colonies. The XS 14 X 3Amgt1i@sstrain appears to be slightly less resistant than other mgtl@ diploids tetrads. Upon dissection, however, the majority of spores were invi (Strains 5â€”7)on the gradient plate, probably due to its slow growth observed with the able. A series of diploids were created in various mutation back XS-l4 haploid. 2717

mgt1@smsh5i@ double mutant failed to do so. As shown in Fig. 2, the All stable recessive MNNG-tolerant revertants (including those relative sensitivity of diploids homozygous for mgtM was the same listed in Table 2) were transformed with plasmid YCp-MSH5 to regardless of the number (0, 1, or 2) of the MSH5 gene copies (Lanes determine whether other independent msh5 mutations result in a 5â€”7).Although the diploid MSHSIXS-l4 was sensitive to MNNG phenotype similar to XS-l4. The MSHS gene was unable to suppress (Lane 8), the corresponding diploid msh5IXS-14 (Lane 9) was clearly MNNG-tolerance in all of the strains examined, nor were other known more resistant than other dipioids studied. These results indicate that MMR genes (data not shown). the phenotype of the diploid is a manifestation of a mutation in the MSHS structural gene. DISCUSSION Second, if the alkylation resistance in XS-l4 is due to a mutation within the MSHS gene and disruption of the MSH5 gene does not The lesion 06 MeG is primarily responsible for the mutagenic and show the same phenotype, we would expect that disruption of the lethal effects of the S@l-type methylating carcinogens (3). Hence, any MSHS gene in XS-l4 results in loss of its MNNG resistant phenotype. hypothesis attempting to explain the genotoxicity of 06 MeG is also Indeed, disruption of the MSHS gene from XS-14 converted it into an relevant to understanding the mechanism of spontaneous and envi MNNG sensitive strain indistinguishable from the 2CmgtM strain ronmental carcinogenesis. The relationship between alkylation toler from which it was isolated (data not shown). ance and MMR as proposed by the abortive MMR hypothesis (6â€”8) Finally, we reasoned that if the msh5â€”14allele from XS-l4 carries is of great interest. First, a significant percentage (20â€”30%)of natu the mutation responsible for the resistant phenotype, it would also rally occurring human tumor cell lines and 60% of SV4O-transformed confer MNNG tolerance in an msh5 deletion strain. We designed cell lines are deficient in MTase activity (MerIMex) and exhibit an experiments using the strategy of targeted integration and position increased sensitivity to killing by methylating agents that produce 06 cloning to isolate the msh5â€”14allele from the XS-l4 mutant. As MeG residues (3, 39). Thus, loss of MTase activity can be a hallmark depicted in Fig. 3A, YIplac2l 1 plasmid carrying the wild-type copy of of tumor development. Second, because eukaryotic cells undergo a the MSHS gene was integrated at the HpaI site upstream of the significant amount of endogenous alkylation damage (40, 41), im genomic msh5â€”14allele. Digestion of the genomic DNA isolated paired MMR would make these cells tolerant of endogenous damage, from the above integrant with HindIlI resulted in a linear molecule thereby conferring a growth advantage, and in turn enhancing spon containing the YIplac2l I vector and the msh5â€”14allele, which was taneous mutagenesis and carcinogenesis. However, the abortive mis recovered by self-ligation and transformation into E. coli cells. The match hypothesis is not conclusive for several reasons. First, the resulting plasmid, YIp-msh5â€”14, was again allowed to integrate into Mer1Mex phenotype of the mammalian cell lines used for estab the 2CmgtMmsh5iX strain by targeted integration at the SnaBI site lishing support of this hypothesis is due to transcriptional down downstream of the msh5@::hisG allele, resulting in Ylplac2l 1 being regulation of, rather than mutations within, the hMGMT gene (42). flanked by msh5@a::hisG and msh5â€”14at the MSH5 locus (Fig. 3B). Actually, the MexIMex@ states in a human lymphoblastoid cell line This chromosome structure was confirmed by Southern hybridization, were found to be interconvertible (43). Second, because alkylation and independent integrants were used for phenotypic analysis. As tolerance is achieved only after several rounds of selection under expected, the 2Cmgt1@ and 2CmgtMmsh5E@ strains were equally mutagenic conditions (8), the phenotype is probably the manifestation sensitive to MNNG-induced killing. However, upon integration of the of more than one mutation. Third, these cell lines were derived from msh5â€”14allele, the 2Cmg:[email protected]@ strain exhibited an alkylation tumors and may carry some preexisting mutations that could contrib tolerant phenotype indistinguishable from the original XS-14 mutant ute to their cellular sensitivity to or tolerance of DNA alkylating (Fig. 3C). agents. Indeed, one of the tolerant mutants (7) was readily revertable

A. C. Hp______SnBH I@w@ II x m.h5- 14

Ion

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______I H ,@ â€”.â€˜ci IIURA3mi544 @SnB MSI15 on Ap

B.. 0 20 40 60 @ Hmah5a:hIaGadâ€” .OiBHI Il@-Iâ€˜cc1 Sn@ Ap UR43mahS-14 Minutes in MNNG Fig. 3. A, strategy for integration and position cloning of the msh5â€”14allele. Plasmid YIp-MSH5, carrying a URA3 marker, was integrated into the XS-14 genome upstream of the msh5â€”14locus by HpaI (Hp) site-directed homologous recombination. The total genomic DNA from the integrant was subjected to Hind!!! (H) digestion and self-ligation, followed by E. coli transformation to recover the plasmid YIp-msh5â€”l4.B,chromosome structure of the 2CmgtMmsh5@./YIp-msh5â€”l4integrantat the MSH5 locus. YIp-msh5â€”l4wasallowed to integrate downstream of the msh5@a::hisGbyprior SnaBl digestion and the integrant structure was confirmed by Southem hybridization. The URA3,Ap, and On genes and multiple cloning sites (triangle) are from the Ylplac2l I vector. C, liquid killing experiment showing MNNG sensitivity of 2C (0). 2Cmgt1@s (1s), [email protected] (R), XS-14 (0), and 2Cmg:[email protected]@./YIp-msh5â€”l4 integrant (â€¢).Cells were treated with 40 @tg/mlMNNG in liquid YPD for the given time. The results are an average of three experiments. 2718

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1997 American Association for Cancer Research. MSH5 MUTATION AND ALKYLATION TOLERANCE to an alkylation-sensitive phenotype. Finally, Koi et al. (44) reported dreds (53) reported that 30% lacked mutations in the coding region of that the alkylation-tolerant human colon cancer cell line HCT1 16, five previously identified MMR genes (hMSH2, hMLHI, hPMSI, which is homozygous for the hmlhl mutation, was rendered sensitive hPMS2, and hMSH6/GTBP). It is therefore anticipated that mutations by transformation with human chromosome 3 carrying a wild-type in additional genes yet to be discovered may act as potential causes of copy of the MUll gene. Dc Wind et a!. (45) reported that transgenic hereditary cancers, and some of them may be new human homologues mice defective in the FZMSH2gene become more resistant to MNNG of MutS, MutL, or other components of the MMR system. We have when endogenous MTase is depleted. In both experiments, however, determined the entire nucleotide sequence of the msh5â€”14coding cells were MTase proficient, and the altered level of alkylation sen region and have identified five nucleotide substitutions, four of which sitivity/resistance after genetic manipulation differs by orders of mag result in amino acid substitutions (Q454R, L498F, L524F, and nitude from those previously reported (7, 8). Y823H). We are in the process of determining which mutation or We have previously demonstrated that null mutation of known combination of mutations is actually responsible for the MNNG MMR genes MLHJ, MSH2, MSH3, and PMSJ did not rescue yeast tolerant phenotype observed in the XS-l4 strain. MTase-deficient cells from killing by DNA methylating agents (23). In this study, we showed that null mutation of two other MSH genes, ACKNOWLEDGMENTS MSH5 and MSH6, also did not confer a MNNG-tolerant phenotype. It is thus concluded that simple loss of MMR activity in yeast is not We thank many laboratories, as listed in Materials and Methods, for pro associated with loss of O@MeG/O4 MeT genotoxicity. However, we viding plasmids and yeast strains. We especially thank Dr. N. Hollingsworth found that a recessive mutation within the MSHS gene, but not the for valuable comments and advice. We also thank Treena Fontanie and Barbara nzsh5i@-nullmutation, is able to make mgtth cells tolerant of MNNG. Chow for technical assistance. The XS-14 cells carrying the msh5â€”14mutationalso display a meiotic defect but not a mutator phenotype. This phenomenon is reminiscent REFERENCES of alkylation-tolerant clones isolated from HeLa cells (46), which also I. Loechler, E. L., Green, C. L., and Essigmann, J. M. In vivo mutagenesis by 0Â°- appear to fall into two groups: one group with a mutator phenotype methylguanine built into a unique site in a viral genome. Proc. NatI. Acad. Sci. 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Sonya Bawa and Wei Xiao

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