Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line

Simon E. Tobi1,4, Dan D. Levy2, Michael M. Seidman11,3 and Kenneth H. Kraemer11,5

1 Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA,
2 CFSAN, Food and Drug Administration, Washington, DC, USA and
3 Laboratory of Molecular Genetics, National Institute of Aging, Baltimore, MD, USA


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12–140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis.


    Introduction
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 Abstract
 Introduction
 Materials and methods
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Studies in Escherichia coli and yeast have shown that postreplication mismatch repair is vital to maintaining the stability of simple repetitive sequences in DNA (1,2). The observation of a high degree of microsatellite instability in the cells from certain human tumors [e.g. hereditary non-polyposis colorectal cancer led to the identification of mutations in several human homologues of yeast mismatch repair proteins, namely MSH2, MLH1, PMS1 and PMS2 (36)]. Apart from causing instability in microsatellite sequences scattered throughout the genome, defects in mismatch repair might also be expected to produce mutations in the repeat motifs that occur within the coding sequences of genes. Several mismatch repair-deficient colon carcinoma cell lines produce multiple single base frameshifts (a mutation hotspot) within a G6 run in the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (79) or a G8 run in the BAX gene (10). Other colon cancer lines have been shown to harbor frameshift mutations in the coding region of the type II transforming growth factor ß receptor, mostly occurring within a mononucleotide A10 run as one or two base deletions (11). Loss of function of this receptor might produce a proliferative advantage leading to malignancy. Subsequent studies have identified two additional human mutS homologues, hMSH6 and hMSH3, each of which purifies as a complex with hMSH2 (1214). In vitro analysis suggests that while the hMSH2–hMSH6 complex (mutS{alpha}) repairs base–base mismatches and single base loop-outs, the hMSH2–hMSH3 complex (mutSß) is directed towards larger loop-out mismatches although recent data point to some degree of redundancy in their roles (15,16).

We examined the effect of the human mismatch repair system on spontaneous mutagenesis in the context of a mononucleotide repeat. We have utilized a shuttle vector system developed in this laboratory (17) based on an E.coli suppresser tRNA, supF, which acts as a highly sensitive mutagenic target for both base substitution and frameshift mutations. A construct was prepared (pLSC) in which the tRNA sequence of supF in pSP189 (18) was modified to contain an interrupted 29 bp mononucleotide poly(G) run. As sequence controls, we made use of a plasmid containing the unmodified supF sequence, pSP189 (18).

Several observations suggest that the mismatch repair system may be involved in the processing of UV damage. Mutations in mutS and mutL of E.coli and in the human hMSH2 and hMLH1 mismatch repair proteins, produce defects in post-UV transcription-coupled repair (19,20). In addition, the human homologue of mutS (hMSH2) has been reported to bind to thymine–thymine dimers and 6–4 thymine–thymine photoproducts (21). We have, therefore, extended our study to examine the effect of DNA mismatch repair on processing of a UV-irradiated shuttle-vector template.

We have studied mutagenesis after replication of the shuttle vectors in the mismatch repair-deficient human lymphoblastoid line, MT1, and the parental mismatch repair-proficient TK6 line (22). The MT1 derivative was first isolated by virtue of its resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (1500-fold) and subsequently reported to show a 60-fold increase in spontaneous mutations at the hprt locus (23). More recently, the mismatch repair defect in the MT1 line has been pinpointed to two mutations in the GT binding protein (GTBP) (24,25), also known as hMSH6 (26), the human homologue of the Saccharomyces cerevisiae MSH6 protein (27). In contrast with MT1 cells, the tumor line HCT-15 (and its genetically related DLD-1 line) which has been the subject of several studies of mutagenesis (7,9,28) contains not only mutated hMSH6 alleles but in addition a point mutation in the polymerase {delta} gene (29,30).

Our results show that, while there was no increase in the frequency of spontaneous mutants produced by the mutant MT1 line in pLSC, the distribution of mutation types was very different from that in the wild-type TK6 line. However, the mismatch repair defect in MT1 cells did not affect UV survival or mutagenesis of the shuttle vector plasmids.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
The human lymphoblastoid cell lines, TK6 and its alkylation-tolerant derivative MT1 (23), (gifts from W.Thilly) were cultured in RPMI 1640 medium supplemented with 16% fetal bovine serum and 2 mM glutamine (Life Technologies, Gaithersburg, MD) at 37°C in a 5% CO2 atmosphere.

Plasmids
pLSC was constructed from pSP189 (DDBJ/EMBL/GenBank accession no. U14594.gb_sy) which was previously constructed in this laboratory (18). Complementary synthetic oligonucleotides were phosphorylated, annealed and ligated into the XhoI–SacI-digested recipient vector (31). The new tRNA and surrounding altered sequence are shown in Figure 1Go. pZ189K (32), a variant of pZ189 with the bacterial gene for kanamycin resistance in place of the ampicillin resistance gene was a gift from Dr Steve Akman (Winston-Salem, NC).



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Fig. 1. The DNA sequence of the tRNA gene and adjacent sequence of pLSC folded into the secondary structure of the mature tRNA product. Bases coding for the mature tRNA product are indicated in uppercase letters; lowercase letters are sequences trimmed at the RnaseP site. Boxes indicate mononucleotide G:C runs. Double-line boxes are bases which were altered from pSP189. Solid lines indicate potential base pairing in the stems of the mature tRNA. Dashed line indicates a potential T:G mispairing. The plasmid sequence beyond XhoI and SacI is identical to pSP189 (18).

 
UV-irradiation and transfection
Cesium chloride-purified plasmid stocks were diluted to a concentration of 31 µg/ml in water and irradiated on ice as described previously (32). Transfections of the plasmids into the human cell lines were performed using DEAE dextran. A UV-irradiated or untreated plasmid sample (1 µg) of pLSC or pSP189 was mixed with 1 µg of unirradiated pZ189K in 1 ml of medium containing 500 µg DEAE dextran and added to 30x106 exponentially growing cells. Following a 10 min incubation at 37°C in a 5% CO2 atmosphere, the cells were washed with 15 ml of fresh medium and resuspended in 40 ml of RPMI 1640 for further culture.

Plasmid recovery, selection of mutants and sequencing
After 48 h in culture, replicated plasmids were recovered from the cells by an alkaline lysis procedure (33) and transformed into an indicator bacterial strain MBM7070 (17) by electroporation (32). Transformation mixtures were plated onto LB-Amp or LB-Kan plates coated with X-gal (20 mg/ml) and IPTG (50 mg/ml). Plasmids containing a mutated, inactive supF tRNA sequence yielded white colonies due to a lack of suppression of the amber codon in the LacZ gene of MBM7070; functional supF produced blue colonies.

Mutant pLSC plasmids containing the mononucleotide poly(G) run were initially sequenced from mini-preps using Sequenase 2.0 (Amersham, Piscataway, NJ) (34). However, cycle sequencing with Thermosequenase (Amersham) using a single-colony lysis method (35) subsequently proved to produce better quality gels more rapidly. pSP189 was also analyzed by cycle sequencing using either Thermosequenase or Sequitherm polymerase (Epicentre Technologies, Madison, WI). Sequencing primers were as follows: for pSP189, the coding strand (forward) primer (5'-GGCGACACGGAAATGTTGAA) (31) was used. Sequencing pLSC with this primer proved difficult through the poly(C) run, but a reverse strand primer (5'-TTTGTGATGCTCGTCAGGGG) provided a clear reading through the poly(G) repeat. Clonal duplicates were excluded by only counting one mutation at a given site per transfection. However, studies with a similar plasmid carrying a random, signature sequence have shown a low frequency of these clonal duplicates (18).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
pLSC mutagenesis
pLSC contains a novel sequence expected to function as a suppressor tRNA in E.coli. It was made by modifying the sequence of supF, a tyrosine suppressor of amber stop codons. As shown in Figure 1Go, modest changes were made in the sequence which codes for two of the tRNA stems. These changes created mononucleotide runs of three, five, seven, nine and 13 [G:C] base pairs. Most of these changes were configured to allow base pairing to occur in the mature tRNA product, although one G:T wobble mispair was included (Figure 1Go). The sequence outside of the mature tRNA was more highly modified. The inserted sequence codes for a functional equivalent of supF, since when transformed into an appropriate E.coli host, the host bacteria were able to metabolize X-gal, through suppression of the amber mutation in the episomal lacZ gene.

The tRNA gene of pLSC was inactivated by variety of base substitution and deletion mutations (see below). In addition to those described, the gene is inactivated by single base insertion mutations in the mononucleotide runs of seven and nine [G:C] pairs. Mutations in the longest run of 13 [G:C] pairs were not expected to alter tRNA function since these lie entirely outside the mature tRNA product.

Spontaneous mutagenesis produced by TK6 and MT1 lines
Mutation frequency
. The frequency of mutant plasmids obtained in pLSC and pSP189 following replication of the untreated plasmids in the TK6 or mismatch repair-deficient MT1 cell lines was similar. Spontaneous pLSC mutants were produced at a frequency of 0.071 ± 0.006% in TK6 cells and 0.077 ± 0.009% in MT1 cells. The spontaneous mutation frequency was lower with pSP189 but was not significantly different between the TK6 (0.025 ± 0.007%) and MT1 (0.017 ± 0.003%) cells.

Analysis of mutations.
Inactivating mutations in the supF gene (producing white or light blue colonies) were characterized by sequencing. Figure 2Go displays the frequency of different classes of spontaneous mutations obtained in pLSC and pSP189 following replication in either TK6 or MT1 cells. The mononucleotide sequence of pLSC provoked a large shift in the classes of mutant plasmids observed in both cell lines with a greater increase in deletions in the repair-deficient cell line. Thus, in the pLSC plasmid, deletion mutations accounted for 41% of all mutations from TK6 cells and 61% from MT1 cells (P = 0.06). Small frameshift mutations were never observed using pSP189, but in pLSC they represented 18% of the mutations in TK6 and almost half of all mutated plasmids (49%) from MT1 cells (P = 0.002).



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Fig. 2. Classes of spontaneous mutations in pLSC and pSP189 replicated in wild-type and mismatch repair-deficient cells. Independent mutant plasmids pLSC (left) and pSP189 (right), isolated from the wild-type (TK6, upper) and mismatch repair-deficient (MT1, lower) cells were sequenced. Plasmids containing the supF marker gene were characterized as having a single base deletion (solid area), 10 or more bases deleted (cross hatched area), a single base substitution (wide bands), two or more base substitutions (narrow bands) or an insertion (open area) in the marker gene. (One deletion **P = 0.002 TK6 versus MT1 for pLSC).

 
The locations of mutations in pLSC are displayed in the spectra of Figure 3Go. In both cell lines, the spontaneous deletions were localized to the mononucleotide poly(G) run. There was a hotspot for single base deletions at position 170 and within the region 171–179 in MT1 cells. Only one insertional event (+GG) was observed (in TK6 cells) in this study; however, our system is sensitive to + frameshifts since passage of pLSC through a MutS E.coli mutant yields a significant number of insertional events in the mononucleotide poly(G) region (data not shown).



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Fig. 3. Location of spontaneous mutations in pLSC and pSP189 replicated in wild-type and mismatch repair-deficient cells. A portion of the modified supF suppressor tRNA marker gene (containing the interrupted mononucleotide G:C run, shaded) in pLSC (A and B) and of the unmodified supF gene in pSP189 (C and D) is shown. Single base deletions are indicated by {Delta}, larger deletions are indicated by a {Delta} at each end connected by a solid line. Base substitutions are indicated below the altered base pair as a change in the lower strand. Each letter represents the mutation found in a sequenced independent plasmid. Tandem mutations (consisting of mutations in adjacent bases or two bases with one intervening base) and multiple base substitutions in one plasmid are indicated by underlining. Data are from experiments presented in Figure 2Go. (A) pLSC replicated in TK6 cells (40 plasmids sequenced). (B) pLSC replicated in MT1 cells (56 plasmids sequenced). (C) pSP189 replicated in TK6 cells (38 plasmids sequenced; two plasmids with large deletions not shown). (D) pSP189 replicated in MT1 cells (40 plasmids sequenced; 1 plasmid with large deletion not shown).

 
In contrast to the deletion mutagenesis occurring in pLSC, no spontaneous single base deletions were found in pSP189 [which lacked the mononucleotide poly(G) run] after replication in either the TK6 or MT1 cells (Figures 2 and 3GoGo). The majority of mutations in both cell lines were single base substitutions. The distributions of spontaneous pSP189 mutations (Figure 3C and DGo) showed no differences between the two cell types except for a T->C hotspot in MT1 cells at position 153 (P = 0.016).

The types of spontaneous single base substitution mutations found in pLSC and pSP189 are displayed in Figure 4Go. Since tandem and multiple base mutations probably occur by a different mechanism in the shuttle vector (36), these were excluded from the analysis. With both plasmids, G:C->A:T transitions were the major base substitutions in TK6 cells [pLSC, 10/15 (67%); pSP189, 18/22 (82%)]. In contrast, this mutation made up a much lower proportion of single base changes in MT1 cells [pLSC, 7/19 (37%); pSP189, 11/33 (33%)—TK6 versus MT1, P <0.005]. A:T->G:C transitions made up 30% of pSP189 single base transitions from MT1 but none was detected from TK6 cells (P = 0.003). The only type of transversion mutation which differed significantly between the two cell lines was a G:C->T:A change in pSP189 (TK6, 1/22; MT1, 8/33; P = 0.05).



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Fig. 4. Types of spontaneous mutations in pLSC and pSP189 replicated in wild-type and mismatch repair-deficient cells. Single base substitution mutations in pLSC (left) and pSP189 (right), isolated from the wild-type (TK6, upper) and mismatch repair-deficient (MT1, lower) cells from experiments in Figure 3Go are shown. G:C->A:T mutations (solid area), A:T->G:C (cross hatched), G:C->T:A (wide bands), G:C->C:G (narrow bands), A:T->T:A (vertical bands) and A:T->C:G (horizontal bands) are indicated. (*P = 0.05, **P <0.004 TK6 versus MT1 for pSP189.)

 
UV plasmid survival
The survival of pLSC and pSP189 following UV-C irradiation of the plasmid and replication in the TK6 and MT1 cell lines is shown in Figure 5AGo. These experiments involved co-transfecting an unirradiated kanamycin plasmid, pZ189K, into the cell lines to act as an internal standard and thus reduce inter-sample variability (32). Survival was reduced in all cases with increasing UV dose. The plasmid survival was slightly greater in the MT1 cells than in the TK6 cells with both pLSC and pSP189.



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Fig. 5 Post-UV plasmid survival and mutation frequency in wild-type and mismatch repair-deficient cells. UV treated pLSC (open symbols) and pSP189 (closed symbols) were transfected into repair-proficient lymphoblastoid cells (TK6, triangles) and its mismatch repair-deficient subline (MT1, circles). In each experiment untreated pZ189kan was cotransfected along with the UV treated plasmid as an internal standard. Replicated plasmids were harvested and used to transform indicator bacteria to ampicillin or kanamycin resistance (32). (A) Relative plasmid survival was measured 2 days after transfection with plasmid treated with 250–1000 J/m2 UV. Each point represents the results of an independent transfection. (B) Frequency of plasmids with mutations after transfection with plasmid treated with 250–1000 J/m2 UV. The proportion of bacterial colonies with mutant plasmids (white or light blue colonies) was compared with those with wild-type plasmids (dark blue colonies). Each point represents the results of an independent transfection [the same transfections as in (A)].

 
UV-induced mutagenesis
The frequency of mutant plasmids recovered from the two cell lines increased with increasing UV doses to the plasmids (Figure 5BGo). The mutation frequencies were higher with pSP189; however, both TK6 and MT1-derived samples contained similar mutant fractions at each UV dose. At 1000 J/m2 UV treatment to the plasmid, there was a 12–21-fold increase in mutation frequency with pLSC and a 74–140-fold increase with pSP189 compared with the untreated plasmids.

UV mutation analysis
Deletion mutations were much less frequent following UV treatment of pLSC plasmids (Figure 6Go) in comparison with the spontaneous frequency (Figure 2Go) with both cell types. Thus, with TK6 cells only 4% (2/52) plasmids contained deletions, and with MT1 only 7% (4/59) plasmids contained deletions. UV treatment of the pLSC produced single base substitutions with the greatest abundance as in earlier studies of normal and excision repair-deficient cells with pSP189 (18) and pZ189 (18,3741). With both cell lines and both plasmids 54–92% of mutant plasmids were single base substitutions (Figure 6Go). Tandem base substitution mutations (involving adjacent bases or bases with 1 intervening base) were the next most abundant (7–25%) for each plasmid, followed by multiple base substitutions (three or more base substitutions or two base substitutions >2 bases apart) (2–15%). There were, however, differences between pLSC and pSP189 in the abundance of plasmids with tandem and multiple mutations. The frequency of tandem mutations was significantly greater in pLSC than in pSP189 for both TK6 (P = 0.03) and MT1 (P = 0.007) cell lines. Plasmids with multiple mutations were also significantly more frequent in pLSC mutants from MT1 cells (compared with pSP189) (P = 0.007) but not in the case of TK6 cells.



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Fig. 6 Classes of post-UV mutations in pLSC and pSP189 replicated in wild-type and mismatch repair-deficient cells. Independent mutant plasmids pLSC (left) and pSP189 (right), isolated from the wild-type (TK6, upper) and mismatch repair-deficient (MT1, lower) cells from experiments in Figure 5Go were sequenced. Plasmids containing the supF marker gene were characterized as having a single base deletion (solid area), 10 or more bases deleted (cross hatched area), a single base substitution (wide bands), two or more base substitutions (narrow bands) or an insertion (open area) in the marker gene. (*P = 0.03 pLSC versus pSP189 for TK6; **P = 0.007 pLSC versus pSP189 for MT1.)

 
The UV mutation spectra for plasmids pLSC and pSP189 are shown in Figure 7Go. There were hotspots for base substitution mutations in pLSC at positions 155 and 159 with TK6 cells and at positions 120 and 159 with MT1 cells. The mononucleotide poly(C) run contains a high frequency of photoproducts (42); however, there were relatively few mutations observed. There were two deletion mutations in the interrupted mononucleotide poly(C) run with MT1 cells, but there were no base substitution mutation hotspots in the poly(C) region beginning at base pair 168. pSP189 mutants recovered from TK6 cells showed the strongest hotspots at positions 155, 156, 164 and 169. The pSP189 mutants in MT1 cells were at positions 156 and 172. There were no significant differences in frequencies of types of transition or transversion mutations between the UV-treated plasmids with either cell line.



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Fig. 7. Location of post-UV mutations in pLSC and pSP189 replicated in wild-type and mismatch repair-deficient cells. A portion of the modified supF suppressor tRNA marker gene (containing the interrupted mononucleotide G:C run, shaded) in pLSC (A and B) and of the unmodified supF gene in pSP189 (C and D) are shown. Base substitutions are indicated below the altered base pair as a change in the lower strand. Each letter represents the mutation found in a sequenced independent plasmid. Tandem mutations (consisting of mutations in adjacent bases or two bases with one intervening base) and multiple base substitutions in one plasmid are indicated by underlining. Single base deletions are indicated by {Delta}, larger deletions are indicated by a {Delta} at each end connected by a solid line. Data are from experiments presented in Figures 5 and 6GoGo. (A) pLSC replicated in TK6 cells (52 plasmids sequenced). (B) pLSC replicated in MT1 cells (60 plasmids sequenced). (C) pSP189 replicated in TK6 cells (68 plasmids sequenced). (D) pSP189 replicated in MT1 cells (62 plasmids sequenced).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
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 References
 
Spontaneous mutation frequency
We did not observe an increase in the frequency of mutant pLSC or pSP189 plasmids recovered from the mismatch repair-defective MT1 cell line despite a reported 60-fold increase in spontaneous mutation frequency at the endogenous hprt locus (23). It is believed that the spontaneous mismatches occur in chromosomal DNA as a consequence of replication errors, which are then repaired in a strand-specific manner. The rate limiting step for mutations which are eliminated by the mismatch repair system is the repair event. Loss of mismatch repair would be noticed as an increase in mutation frequency, particularly at runs of simple sequence where mismatches are most likely to appear.

However, in the shuttle vector, the basis of spontaneous mutations is not replication. Most spontaneous mutations are caused early in transfection prior to vector replication and the frequency of spontaneous vector mutations is higher than the frequency of spontaneous mutations in a chromosomal marker. This is because the vector is exposed to damaging events, such as imposed by nicking activities, that are substantially more frequent than seen by a chromosomal marker. And that is probably because the vectors enter the cell as naked DNA, rather than as chromatinized DNA. There are several consequences to the nick: deletions, insertions, point mutations, multple point mutations, etc. When we provide the opportunity for some slippage we add another kind of mutational event to the mix. However, there is already an appreciable level of mutagenesis. So it is likely that the spontaneous mutation frequency did go up a little but not by enough to notice.

Mutations in the supF marker gene of the shuttle vector plasmid do not alter plasmid survival and, thus, are not selected for or against. The mutagenic target in the shuttle vector is the gene that codes for tRNA, where most bases are essential (43), rather than one that codes for protein such as hprt, where the third base in each codon (the wobble position) may be changed without altering protein function. Furthermore, the functioning of the supF gene is not essential for plasmid survival. Thus, the plasmid system may be more able to detect mutations than the hprt system. This may contribute to a higher spontaneous mutation frequency in the shuttle vector compared with a chromosomal gene (79) and a failure to see increased spontaneous mutations in the mismatch repair-deficient MT1 cells.

Spontaneous deletion mutations
In order to study the impact of loss of hMSH6 function on mutagenesis in human cells, we have modified the supF gene to contain runs of mononucleotides [poly(G):C], producing a target sensitive to both frameshift and base substitution mutagenesis (pLSC). Our data demonstrate a clear shift to spontaneous single base deletion mutations in the interrupted mononucleotide poly(G) run of pLSC by replication in a hMSH6 mutant background (MT1).

Naked plasmid DNA transfected into cells is nicked by cellular processes. The nick gives the region an opportunity to be replicated by an error-prone polymerase that can make single point mutations but can also give rise to the slippage product of a mismatch (loop-out). In mismatch repair-proficient cells this is recognized and repaired, in mismatch repair-deficient cells they are not repaired, since they are localized to the mononucleotide poly(G) runs and that is where we see them. If we eliminated all the scattered point mutations and the large deletion mutations from the spontaneous spectrum (making the collection more like a chromosomal collection) then our data indicates a large increase in spontaneous single base deletions in the mismatch repair-deficient cells (Figure 2Go).

When considering the specific single base deletion frameshift mutation the frequency is nearly 3-fold greater in the MT1 cell line than in the TK6 with pLSC. Data from S.cerevisiae indicate that the mutator phenotype produced by loss of MSH6 function is weak compared with that of MSH2 mutants both in the context of a G18 repeat (32- versus 6300-fold over wild-type) and in a target (CAN1) prone mainly to base substitutions (7- versus 32-fold over wild-type) (44). As a chromosomally integrated sequence in {lambda} phage, however, supF does reflect a mutator phenotype in mice deficient in PMS2 (45). Our results are consistent with the findings that hMSH6/GTBP mutants are not as strong mutators as hMSH2 mutants, perhaps because of the ability of hMSH3 to bind to hMSH2 and partially complement the defect in hMSH6 (15). Clearly this complementation is only partial since there are definite repair deficiencies in hMSH6 cell lines.

Sequence effects on mutations
Comparison of the spontaneous mutation spectra obtained from pLSC and pSP189 suggests that a mononucleotide run of G or C longer than the five in the latter plasmid (positions 172–176) is required to produce deletion mutations. However, the sequence context of a repeat may also be important since the 7 base mononucleotide stretch from 99 to 105 in pLSC shows little deletion activity in our cell lines. In addition, it should be noted that there is a single sequence difference between the two targets at position 171 which might affect mutagenesis. The mononucleotide poly(G) run of pLSC tends to be a stronger focus for mutations in the mismatch repair-deficient MT1 cells than in the TK6 parental line [75% (45/60) of all mutations in MT1 and 43% (23/53) of all mutations in TK6 are in the shaded repeat region]. This is consistent with data in the yeast S.cerevisiae which point to mononucleotide runs as strong targets for mutagenesis in mismatch repair-deficient cells (46,47). Moreover, analysis of human colonic tumors deficient in mismatch repair has shown that mononucleotide tracts are preferentially targeted in genes whose inactivation may be important for tumorigenesis, e.g. a G8 stretch of the proapoptotic gene BAX (10,48,49), an A10 run in the type II transforming growth factor receptor gene (11), and the APC gene (50).

The predominance of single deletions in the poly(G):C tract of pLSC mutants from MT1 cells is consistent with a role for the hMSH6–hMSH2 complex (mutS{alpha}) in the repair of single insertion/deletion mismatches. Indeed, cell extracts from MT1 cells are defective in repair of single nucleotide—loop-out—heteroduplexes as well as base–base mismatches; in contrast, repair of larger (two, three or four) insertion/deletions is relatively efficient (12). This selectivity in repair is borne out by the observed instability of a poly(A) repeat marker but not a dinucleotide (CA) microsatellite from these cells (25). However, mononucleotide runs tend to produce only deletions of a single base in several mismatch mutant backgrounds where the defects (e.g. MSH2 and PMS2 ) would be expected to extend to loop-outs of two or even three bases (4446). These findings suggest that slippages of only one base occur during replication of such a repeat if the model of (51) is invoked. The lack of insertions in our spectrum from MT1 cells is also in broad agreement with the predominance of deletions in mononucleotide stretches in mismatch deficient yeast (44,46). The supF target is clearly sensitive to this event, however, since a number appear in the PMS2 background of the study by Narayanan (45). The appearance of larger deletions of >=13 bases in the spectra from wild-type TK6 cells suggests that even a functional mismatch repair system cannot efficiently correct errors in all types of repetitive DNA. It is possible that the replication machinery is confronted with topological challenges other than simple strand slippage during processing of such sequences. In any case, although localized to the mononucleotide poly(G) run of pLSC, these deletions may not be the result of unrepaired loop-outs but may arise through a different mechanism.

hMSH6 mutations and cancer
Mutations in hMSH6 have been found in several solid tumor (25,52) and leukemia lines (53). There is a report of one hereditary non-polyposis colon cancer family with a germline mutation in hMSH6 (48). However, mice carrying a null mutation in MSH6 do develop spontaneous gastrointestinal tumors and lymphomas without microsatellite instability (54).

hMSH6 deficiency and UV repair
Escherichia coli strains that are defective in the DNA mismatch repair genes mutS and mutL are moderately sensitive to killing by UV radiation and are unable to perform transcription-coupled excision repair (20). Similarly, human tumor cell lines deficient in one of three mismatch repair genes (hMSH2, hPMS2 or hMLH1) were slightly hypersensitive to killing by UV and showed deficiency of transcription-coupled DNA repair (19). Deficiency in transcription-coupled DNA repair is seen in cells from patients with the hereditary, progressive disorder, Cockayne syndrome (55). We previously found that defective transcription-coupled DNA can be detected by the plasmid shuttle vector in cells from patients with Cockayne syndrome as decreased post-UV plasmid survival and increased post-UV plasmid mutability (34). However, in the present study there was similar post-UV plasmid survival and post-UV plasmid mutation frequency in the mismatch repair-deficient (MT1) and proficient (TK6) cells. This suggests that hMSH6 deficient cells (MT1) are proficient in transcription coupled DNA repair.

The interrupted mononucleotide sequence in pLSC containing C25 on one strand (and G25 on the other) is expected to be a region of intense UV-induced DNA damage in the polypyrimidine tract (42) as well as being subject to creation of small loops that are subject to base slippage. The hMSH2 protein binds to cissyn thymine–thymine cyclobutane dimers and 6–4 thymine–thymine dimers (21) while the hMSH2–hMSH6 complex binds too weakly to these lesions (56). However, there were only a few base substitution mutations (mostly CC to TT tandems) found in this region with the UV-treated plasmid pLSC in the mismatch repair-deficient or proficient cells. These UV-treated mononucleotide regions are potential sites for compound DNA lesions (base damage and mismatch) of the type studied by Mu et al. (56). Our finding of no apparent effect of human mismatch repair on UV damage processing is in keeping with the results of Mu et al. (56), in which no functional overlap between excision repair and mismatch repair was observed.

Only three frameshift mutations (two single base deletions and one single base insertion in the MT1 cells) were seen with the UV treated pLSC (Figure 7Go). In the plasmid pSP189 there is a shorter mononucleotide sequence of C5 that was a hotspot for C->T transversion mutations in the mismatch repair-deficient MT1 cells. However, there were no frameshift mutations in this region with UV treated pSP189. Nucleotide excision repair creates an ~30 bp gap during processing of UV damage (57). Since there must be extensive repair of the C run in the UV-treated plasmid, the repair synthesis step must be quite unlikely to generate mismatches. Thus, the structure of the gap filling complex must preclude formation of mismatched sequences, at least those that would require hMSH6 mismatch repair activity to remove.


    Notes
 
4 Present address: Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK Back

5 To whom correspondence should be addressed Email: kraemerk{at}nih.gov Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Modrich,P. and Lahue,R. (1996) Mismatch repair in replication fidelity, genetic recombination and cancer biology. Annu. Rev. Biochem., 65, 101–133.[ISI][Medline]
  2. Strand,M., Prolla,T.A., Liskay,R.M. and Petes,T.D. (1993) Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature, 365, 274–276.[ISI][Medline]
  3. Fishel,R., Lescoe,M.K., Rao,M.R., Copeland,N.G., Jenkins,N.A., Garber,J., Kane,M. and Kolodner,R. (1993) The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer [published erratum appears in Cell (1994) 77, p. 167]. Cell, 75, 1027–1038.
  4. Leach,F.S., Nicolaides,N.C., Papadopoulos,N., Liu,B., Jen,J., Parsons,R., Peltomaki,P., Sistonen,P., Aaltonen,L.A. and Nystrom-Lahti,M. (1993) Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell, 75, 1215–1225.[ISI][Medline]
  5. Papadopoulos,N., Nicolaides,N.C., Wei,Y.-F. et al. (1994) Mutation of a mutL homolog in hereditary colon cancer. Science, 263, 1625–1629.[ISI][Medline]
  6. Nicolaides,N.C., Papadopoulos,N., Liu,B. et al. (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature, 371, 75–80.[ISI][Medline]
  7. Malkhosyan,S., McCarty,A., Sawai,H. and Perucho,M. (1996) Differences in the spectrum of spontaneous mutations in the hprt gene between tumor cells of the microsatellite mutator phenotype. Mutat. Res. DNAging Genet. Instability Aging, 316, 249–259.
  8. Bhattacharyya,N.P., Ganesh,A., Phear,G., Richards,B., Skandalis,A. and Meuth,M. (1995) Molecular analysis of mutations in mutator colorectal carcinoma cell lines. Hum. Mol. Genet., 4, 2057–2064.[Abstract]
  9. Ohzeki,S., Tachibana,A., Tatsumi,K. and Kato,T. (1997) Spectra of spontaneous mutations at the hprt locus in colorectal carcinoma cell lines defective in mismatch repair. Carcinogenesis, 18, 1127–1133.[Abstract]
  10. Rampino,N., Yamamoto,H., Ionov,Y., Li,Y., Sawai,H., Reed,J.C. and Perucho,M. (1997) Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science, 275, 967–969.[Abstract/Free Full Text]
  11. Markowitz,S., Wang,J., Myeroff,L., Parsons,R., Sun,L., Lutterbaugh,J., Fan,R.S., Zborowska,E., Kinzler,K.W. and Vogelstein,B. (1995) Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability [see comments]. Science, 268, 1336–1338.[ISI][Medline]
  12. Drummond,J.T., Li,G.-M., Longley,M.J. and Modrich,P. (1995) Isolation of an hMSH2-p160 heterodimer that restores DNA mismatch repair to tumor cells. Science, 268, 1909–1912.[ISI][Medline]
  13. Palombo,F., Iaccarino,I., Nakajima,E., Ikejima,M., Shimada,T. and Jiricny,J. (1996) hMutSß, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion leaps in DNA. Curr. Biol., 6, 1181–1184.[ISI][Medline]
  14. Acharya,S., Wilson,T., Gradia,S., Kane,M.F., Guerrette,S., Marsischky, G.T., Kolodner,R. and Fishel,R. (1996) hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6. Proc. Natl Acad. Sci. USA, 93, 13629–13634.[Abstract/Free Full Text]
  15. Umar,A., Risinger,J.I., Glaab,W.E., Tindall,K.R., Barrett,J.C. and Kunkel,T.A. (1998) Functional overlap in mismatch repair by human MSH3 and MSH6. Genetics, 148, 1637–1646.[Abstract/Free Full Text]
  16. Genschel,J., Littman,S.J., Drummond,J.T. and Modrich,P. (1998) Isolation of MutSß from human cells and comparison of the mismatch repair specificities of MutSß and MutS{alpha}. J. Biol. Chem., 273, 19895–19901.[Abstract/Free Full Text]
  17. Seidman,M.M., Dixon,K., Razzaque,A., Zagursky,R.J. and Berman,M.L. (1985) A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells. Gene, 38, 233–237.[ISI][Medline]
  18. Parris,C.N. and Seidman,M.M. (1992) A signature element distinguishes sibling and independent mutations in a shuttle vector plasmid. Gene, 117, 1–5.[ISI][Medline]
  19. Mellon,I., Rajpal,D.K., Koi,M., Boland,C.R. and Champe,G.N. (1996) Transcription-coupled repair deficiency and mutations in human mismatch repair genes. Science, 272, 557–560.[Abstract]
  20. Mellon,I. and Champe,G.N. (1996) Products of DNA mismatch repair genes mutS and mutL are required for transcription-coupled nucleotide-excision repair of the lactose operon in Escherichia coli. Proc. Natl Acad. Sci. USA, 93, 1292–1297.[Abstract/Free Full Text]
  21. Fishel,R. and Wilson,T. (1997) MutS homologs in mammalian cells. Curr. Opin. Genet. Dev., 7, 105–113.[ISI][Medline]
  22. Goldmacher,V.S., Cuzick,R.A. Jr and Thilly,W.G. (1986) Isolation and partial characterization of human cell mutants differing in sensitivity to killing and mutation by methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine. J. Biol. Chem., 261, 12462–12471.[Abstract/Free Full Text]
  23. Kat,A., Thilly,W.G., Fang,W., Longley,M.J., Li,G.-M. and Modrich,P. (1993) An alkylation-tolerant, mutator human cell line is deficient in strand-specific mismatch repair. Proc. Natl Acad. Sci. USA, 90, 6424–6428.[Abstract]
  24. Palombo,F., Gallinari,P., Iaccarino,I., Lettieri,T., Hughes,M., D'Arrigo,A., Truong,O., Hsuan,J.J. and Jiricny,J. (1995) GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. Science, 268, 1912–1914.[ISI][Medline]
  25. Papadopoulos,N., Nicolaides,N.C., Liu,B., Parsons,R., Lengauer,C., Palombo,F., D'Arrigo,A., Markowitz,S., Willson,J.K.V., Kinzler,K.W., Jiricny,J. and Vogelstein,B. (1995) Mutations of GTBP in genetically unstable cells. Science, 268, 1915–1917.[ISI][Medline]
  26. Iaccarino,I., Marra,G., Palombo,F. and Jiricny,J. (1998) hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutS{alpha}. EMBO J., 17, 2677–2686.[Abstract/Free Full Text]
  27. Iaccarino,I., Palombo,F., Drummond,J., Totty,N.F., Hsuan,J.J., Modrich,P. and Jiricny,J. (1996) MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2. Curr. Biol., 6, 484–486.[ISI][Medline]
  28. Glaab,W.E. and Tindall,K.R. (1997) Mutation rate at the hprt locus in human cancer cell lines with specific mismatch repair-gene defects. Carcinogenesis, 18, 1–8.[Abstract]
  29. Da Costa,L.T., Liu,B., El-Deiry,W.S., Hamilton,S.R., Kinzler,K.W., Vogelstein,B., Markowitz,S., Willson,J.K.V., De la Chapelle,A., Downey,K.M. and So,A.G. (1995) Polymerase {delta} variants in RER colorectal tumours. Nature Genet., 9, 10–11.[ISI][Medline]
  30. Umar,A., Koi,M., Risinger,J.I., Glaab,W.E., Tindall,K.R., Kolodner,R.D., Boland,C.R., Barrett,J.C. and Kunkel,T.A. (1997) Correction of hypermutability, N-methyl-N'-nitro-N-nitrosoguanidine resistance and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6. Cancer Res., 57, 3949–3955.[Abstract]
  31. Parris,C.N., Levy,D.D., Jessee,J. and Seidman,M.M. (1994) Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells. J. Mol. Biol., 236, 491–502.[ISI][Medline]
  32. Moriwaki,S.-I., Tarone,R.E. and Kraemer,K.H. (1994) A potential laboratory test for dysplastic nevus syndrome: Ultraviolet hypermutability of a shuttle vector plasmid. J. Invest. Dermatol., 103, 7–12.[Abstract]
  33. Parris,C.N. and Kraemer,K.H. (1992) Ultraviolet mutagenesis in human lymphocytes: the effect of cellular transformation. Exp. Cell Res., 201, 462–469.[ISI][Medline]
  34. Parris,C.N. and Kraemer,K.H. (1993) Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal. Proc. Natl Acad. Sci. USA, 90, 7260–7264.[Abstract]
  35. Levy,D.D., Magee,A.D. and Seidman,M.M. (1996) Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots. J. Mol. Biol., 258, 251–260.[ISI][Medline]
  36. Seidman,M.M., Bredberg,A., Seetharam,S. and Kraemer,K.H. (1987) Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity. Proc. Natl Acad. Sci. USA, 84, 4944–4948.[Abstract]
  37. Seetharam,S., Kraemer,K.H., Waters,H.L. and Seidman,M.M. (1991) Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts. Mutat. Res., 254, 97–105.[ISI][Medline]
  38. Seetharam,S., Waters,H.L., Seidman,M.M. and Kraemer,K.H. (1989) Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome. Cancer Res., 49, 5918–5921.[Abstract]
  39. Seetharam,S., Protic-Sabljic,M., Seidman,M.M. and Kraemer,K.H. (1987) Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum. J. Clin. Invest., 80, 1613–1617.[ISI][Medline]
  40. Bredberg,A., Kraemer,K.H. and Seidman,M.M. (1986) Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells. Proc. Natl Acad. Sci. USA, 83, 8273–8277.[Abstract]
  41. Waters,H.L., Seetharam,S., Seidman,M.M. and Kraemer,K.H. (1993) Ultraviolet hypermutability of a shuttle vector propagated in xeroderma pigmentosum variant cells. J. Invest. Dermatol., 101, 744–748.[Abstract]
  42. Brash,D.E., Seetharam,S., Kraemer,K.H., Seidman,M.M. and Bredberg,A. (1987) Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc. Natl Acad. Sci. USA, 84, 3782–3786.[Abstract]
  43. Kraemer,K.H. and Seidman,M.M. (1989) Use of supF, an Escherichia coli tyrosine suppressor tRNA gene, as a mutagenic target in shuttle-vector plasmids. Mutat. Res., 220, 61–72.[ISI][Medline]
  44. Sia,E.A., Kokoska,R.J., Dominska,M., Greenwell,P. and Petes,T.D. (1997) Microsatellite instability in yeast: Dependence on repeat unit size and DNA mismatch repair genes. Mol. Cell. Biol., 17, 2851–2858.[Abstract]
  45. Narayanan,L., Fritzell,J.A., Baker,S.M., Liskay,R.M. and Glazer,P.M. (1997) Elevated levels of mutation in multiple tissues of mice deficient in the DNA mismatch repair gene Pms2. Proc. Natl Acad. Sci. USA, 94, 3122–3127.[Abstract/Free Full Text]
  46. Tran,H.T., Keen,J.D., Kricker,M., Resnick,M.A. and Gordenin,D.A. (1997) Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants. Mol. Cell. Biol., 17, 2859–2865.[Abstract]
  47. Greene,C.N. and Jinks-Robertson,S. (1997) Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins. Mol. Cell. Biol., 17, 2844–2850.[Abstract]
  48. Miyaki,M., Konishi,M., Tanaka,K., Kikuchi-Yanoshita,R., Muraoka,M., Yasuno,M., Igari,T., Koike,M., Chiba,M. and Mori,T. (1997) Germline mutation of MSH6 as the cause of hereditary nonpolyposis colorectal cancer. Nature Genet., 17, 271–272.[ISI][Medline]
  49. Yamamoto,H., Sawai,H., Weber,T.K., Rodriguez-Bigas,M.A. and Perucho,M. (1998) Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer. Cancer Res., 58, 997–1003.[Abstract]
  50. Huang,J., Papadopoulos,N., McKinley,A.J., Farrington,S.M., Curtis,L.J., Wyllie,A.H., Zheng,S., Willson,J.K.V., Markowitz,S.D., Morin,P., Kinzler,K.W., Vogelstein,B. and Dunlop,M.G. (1996) APC mutations in colorectal tumors with mismatch repair deficiency. Proc. Natl Acad. Sci. USA, 93, 9049–9054.[Abstract/Free Full Text]
  51. Streisinger,G., Okada,Y., Emrich,J., Newton,J., Tsugita,A., Terzaghi,E. and Inouye,M. (1966) Frameshift mutations and the genetic code. This paper is dedicated to Professor Theodosius Dobzhansky on the occasion of his 66th birthday. Cold Spring Harb. Symp. Quant. Biol., 31, 77–84.[ISI][Medline]
  52. Risinger,J.I., Umar,A., Boyd,J., Berchuck,A., Kunkel,T.A. and Barrett,J.C. (1996) Mutation of MSH3 in endometrial cancer and evidence for its functional role in heteroduplex repair. Nature Genet., 14, 102–105.[ISI][Medline]
  53. Hosoya,N., Hangaishi,A., Ogawa,S., Miyagawa,K., Mitani,K., Yazaki,Y. and Hirai,H. (1998) Frameshift mutations of the hMSH6 gene in human leukemia cell lines. Jpn. J. Cancer Res., 89, 33–39.[ISI][Medline]
  54. Edelmann,W., Yang,K., Umar,A. et al. (1997) Mutation in the mismatch repair gene Msh6 causes cancer susceptibility. Cell, 91, 467–477.[ISI][Medline]
  55. Bootsma,D., Kraemer,K.H., Cleaver,J.E. and Hoeijmakers,J.H.J. (1998) Nucleotide excision repair syndromes: xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. In Vogelstein,B. and Kinzler,K.W. (eds) The Genetic Basis of Human Cancer. McGraw-Hill, New York, NY, pp. 245–274.
  56. Mu,D., Tursun,M., Duckett,D.R., Drummond,J.T., Modrich,P. and Sancar,A. (1997) Recognition and repair of compound DNA lesions (base damage and mismatch) by human mismatch repair and excision repair systems. Mol. Cell. Biol., 17, 760–769.[Abstract]
  57. Sancar,A. (1996) DNA excision repair. Annu. Rev. Biochem., 65, 43–81.[ISI][Medline]
Received November 18, 1998; revised February 9, 1999; accepted March 17, 1999.