Human Molecular Genetics, 2002, Vol. 11, No. 6 707-713
© 2002 Oxford University Press
Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells
1Department of Pathology and Laboratory Medicine, 2Curriculum in Genetics and Molecular Biology and 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Received November 28, 2001; Revised and Accepted January 20, 2002.
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ABSTRACT |
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We have measured the mutation rates of G17 and A17 repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)17 repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A17 and (CA)17 sequences were similar in the mismatch repair proficient cells, but the mutation rate of G17 was significantly higher than that of either A17 or (CA)17. In the mismatch repair deficient cells, the G17 and (CA)17 mutation rates were similar and were significantly higher than the A17 rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A17 sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G17 sequence. These results indicate that a G17 repeat is less stable than an A17 repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G17 repeats.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many microsatellites, or simple sequence repeats, are polymorphic in the human population, in that there are differences in the numbers of repeats among alleles. Mononucleotide repeats, especially A-rich microsatellites, are abundant in the genomes of humans and other primates (1–3). Instability in microsatellites has been attributed to a DNA polymerase slippage mechanism first proposed by Streisinger et al. (4). These sequences are exceptionally unstable in cells lacking mismatch repair. This instability is evident in tumors of patients with hereditary non-polyposis colorectal cancer (HNPCC) and in the 10–15% of sporadic colorectal and endometrial cancers that are mismatch repair deficient (5,6).
There are a number of properties of microsatellites that may affect their mutation rate: (i) length of the repeat unit (e.g. mono- versus dinucleotides), (ii) base composition of the repeat, (iii) number of repeat units per tract, (iv) degree of perfection of repeats (i.e. presence or absence of interruptions in the tract) and (v) composition of flanking sequences. We report here on the effect of base composition of the repeat on the mutation rate of moderately sized mononucleotide repeats in mammalian cells. It has been demonstrated in the yeast Saccharomyces cerevisiae that a poly-G mononucleotide repeat is more unstable than sequences with larger repeat units (7). We have compared the mutation rates of mononucleotide repeats with those previously determined for dinucleotide repeats in both mismatch repair proficient (MMR+) and mismatch repair deficient (MMR–) cultured cells. We find that a G17 sequence is significantly more unstable than A17 or (CA)17 sequences in cells proficient in MMR; this finding is similar to those recently reported in yeast (8) and Escherichia coli (9). In MMR– cells, where the mutation rates were very high and approach the maximal limit of this assay, G17 and (CA)17 repeat mutation rates were similar and were 
10-fold higher than the A17 mutation rate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have measured the mutation rates of two mononucleotide microsatellite sequences, A17 and G17, and compared these rates to that of the dinucleotide sequence (CA)17, in MMR+ and MMR– cells. Microsatellite sequences were cloned into a plasmid vector, pCon
(Fig. 1A). The plasmids contain a fusion gene consisting of the herpes simplex virus thymidine kinase gene (tk) fused at its 3' end to the 5' end of a bacterial gene coding for neomycin resistance (neo). The microsatellites were inserted near the 3' end of the tk gene, such that the neo gene is in the (–1) reading frame. The sequences of the microsatellite-containing inserts are shown in Figure 1B. Plasmids were transfected into cells, and clones with the plasmid DNA stably integrated into the genome were selected using hygromycin. Cells with frameshift mutations in the microsatellite that restored the reading frame of the neo gene were selected in G418. Fluctuation analyses were performed on three to six transfected clones from each cell line, and mutation rates were calculated by the Luria–Delbrück method of the mean (10).
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H6, a clonal derivative of the human colorectal cancer cell line HCT116 (11), which is deficient in hMLH1 (12), was used for studies of microsatellite mutation rates in MMR– cells. Two MMR+ cell lines were used: CAK is a spontaneously immortalized non-tumorigenic near-diploid mouse cell line, and hTERT1604 is a line of human lung fibroblasts that has been immortalized by telomerase expression. Prior to the availability of the telomerase-immortalized human cells, we used the CAK cells (13) and a strain of normal human foreskin fibroblasts, NHF1, as MMR+ cells (14). NHF1, with its limited lifespan in culture, is difficult to use in fluctuation tests, which require extensive culture expansion; however, the immortal hTERT1604 cells can be analyzed easily and have recently been shown in our laboratory to have mutation rates similar to those of the NHF1 cells for the (CA)17 microsatellite sequence (15). The CAK cells have been included in this study as an additional MMR+ line within which to compare the mutation rates of mononucleotide and dinucleotide repeats.
Two slightly different microsatellite inserts containing G17 sequences were used. As shown in Figure 1B, the G17 and RG17 sequences were identical except for the sequences flanking the mononucleotide repeat. Mutation rates of these two sequences were compared in CAK cells and were found to be very similar. Since the differences in flanking sequences between the two constructs did not affect the mutation rates of the microsatellite, the G17 data were used for comparisons with those of the A17 and (CA)17 microsatellites in hTERT1604 and H6 cells.
Table 1 shows mutation rate data from all of the sequences in each cell line. Mutation rates of individual experiments are shown, as well as the medians of these rates for each cell type. For all three microsatellite sequences, the mutation rates in the MMR– cells were at least two orders of magnitude higher than in the human fibroblasts. The mouse cells (CAK) generally exhibited mutation rates intermediate between that of the hTERT1604 cells and the H6 cells.
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The mutation rates of the A17 and (CA)17 sequences were similar to each other when compared within each of the MMR+ cell lines. Only maximum rate estimates for A17 in hTERT1604 cells could be determined, since no mutants were recovered in experiments with three independent transfected clones; however, we were able to demonstrate that at least two of these clones were capable of producing neoR revertants after treatment with ICR170 (unpublished data), which is a known frameshift mutagen (16). The mutation rates of the G17 sequence in the MMR+ cell lines were much higher than the rates of the A17 or (CA)17 sequences. The differences between the G17 rate and the rates for A17 and (CA)17 in hTERT1604 and CAK were 17-fold or higher and 11- to 20-fold, respectively. In the MMR– H6 cells, the G17 and (CA)17 sequences had similar rates, which were seven to 15 times higher than that of the A17 sequence. The rate difference between G17 and A17 in H6 was statistically significant (P < 0.0001). Note that the H6 data are more difficult to interpret, because the variation among hygR clones tends to be high in these cells, and the mutation rates approached the upper limit of the assay (3 x 10–2 mutations/cell/generation) (14). Overall, the G17 repeat is much more unstable than the A17 repeat in both MMR+ and MMR– cells.
DNA was isolated from G418R clones and PCR was performed on the microsatellite for characterization of the mutations in the mononucleotide repeats. PCR products were analyzed either by autoradiography of radioactively labeled products or by capillary electrophoresis of fluorescently labeled products. Figure 2 is an example of data analyzed using capillary electrophoresis. PCR product profiles of DNA from hygR control cells were compared to those of mutant G418R cells. PCR product profiles exhibit multiple peaks resulting from the tendency of Taq polymerase to produce products of various sizes as a result of slipped-strand pairing during polymerization (17). Shifts in the entire cluster of peaks indicate the size differences between control and mutant DNAs. Table 2 shows the types of frameshift mutations that were detected and their relative frequencies in each experiment. The repeat tracts in the parental cells were all in the –1 reading frame with respect to the downstream neo coding region. The majority of mutations were either +1 or –2 bp changes, either of which would be expected to restore the neo reading frame. These results are consistent with the observation made by us and others that the smallest mutations in repetitive templates are the most common (7,18).
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In the MMR+ cells, +1 mutations predominated for both the A17 and G17 templates. The pattern was different in the MMR– cells. In the H6 cells, mutants of the A17 sequence were mostly deletions of 2 bp, while the G17 sequence produced approximately equal numbers of –2 and +1 mutations. These distributions of types of mutations were significantly different between hTERT1604 and H6 cells (P < 0.0006, Fisher’s exact test). The basis for this difference is not known, but the results of the comparison indicate that the efficiency of MMR on 2-base loops in the template strand in the G17 repeat is highly efficient (99.9%) relative to the efficiency of MMR on 1-base loops in the primer strand (98.3%). Although single base insertions also predominated in the MMR+ CAK cells, the difference in the distributions of mutation types between these cells and the MMR– H6 cells was not significant (P = 0.34).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Repetitive DNA sequences are particularly prone to frameshift mutations, probably as the result of strand slippage during DNA replication (4). These frameshift mutations usually involve insertion or deletion of one or more repeat units. One factor that influences repeat instability is repeat unit size; for example, mononucleotides are the most unstable of the simple repeat sequences in yeast (7).
In humans and primates, mononucleotide repeats are more abundant than all other repeat motifs (2,3). A growing list of genes associated with some types of cancer contain mononucleotide repeats in their coding regions, as discussed below. The instability of these sequences has been observed in tumors from patients with HNPCC (19,20), as well as sporadic colorectal (19,20), gastric (21) and endometrial (22) cancers.
We have used a reversion assay to measure the mutation rates of A17 and G17 mononucleotide repeats and have compared these rates to those of a (CA)17 dinucleotide repeat in order to determine how the nucleotide composition of a repeat can affect its instability. We have also studied the effects of mismatch repair on the stability of these sequences. We found that the G-repeat was more unstable than the A-repeat in both human and mouse cells and that, in the MMR+ cells, the mutation rate of the A17 microsatellite was similar to that of the (CA)17 repeat. The G17 sequence was 10–25-fold more unstable than A17 or (CA)17, depending upon the cell line. In the MMR– H6 cells, the G17 and (CA)17 mutation rates were similar and were 7–15-fold higher than the A17 mutation rate; thus, the relative instabilities of the G17 and A17 repeats were independent of MMR status, although MMR– cells had higher mutation rates for all repetitive templates. The observation that mutation rates in CAK were higher than those in hTERT1604, even though both lines are MMR+, may result from the presence of mutations in other genes in CAK that could lead to greater microsatellite instability or, alternatively, may be the result of the species difference (23).
These results on mononucleotide repeats are consistent with results of similar studies in E.coli (9,24) and yeast (8). Sagher et al. (9) reported that a run of 8 Cs had frameshift mutation rates 12 and 36 times greater than a similar A8 run in wild-type and mutS E.coli, respectively. In wild-type and mlh1 yeast, G10 and C10 runs were approximately three to 19 times more unstable than A10 and T10 runs (8). The hprt locus in MMR– human colon cancer lines exhibits a frameshift hotspot at a run of six Gs (25); however, in a spleen necrosis virus-based vector system, which lacks MMR, the opposite was true: A- or T-runs mutated more frequently than G- or C-runs (26). This result suggests that slippage on an RNA homopolymer template by reverse-transcriptase may be different from replication slippage on mononucleotide sequences by a DNA polymerase. To explain the effect of the base composition of the repeat on the mutation rate of mononucleotide runs, Sagher et al. (9) suggested that the looped out mutational intermediate for a frameshift mutation is more stable in G- or C-runs than in A- or T-runs. Such greater loop stability could result from stronger stacking interactions among Gs or Cs than among As or Ts (9). Others have suggested that unusual DNA structures in homopolymeric sequences may affect slippage rates (27).
The relative abundance of A-repeats and G-repeats in genomes of various taxa may reflect their proposed origins, functions and mutation rates. A-rich sequences are the most abundant of all microsatellites in primates, with CA-repeats the next most frequent. G-runs, on the other hand, occur at a much lower frequency than either of the above (2,3). Most taxa studied exhibit this pattern, with the exception of rodents, in which dinucleotide repeats are most abundant. Analysis of genomic context of repetitive sequences has shown that many A-rich sequences are contiguous to the 3' ends of retroposons (Alu and Line-1), which may have contributed to their genomic-wide abundance (2,3). In addition, it has been suggested that A-rich microsatellites play a role in organization of eukaryotic chromatin, as they are found in matrix-associated sequences (2).
The paucity of G-repeats in most genomes may reflect their higher mutation rates. The mutational tendencies of these sequences have provided pathogenic bacteria with a strategy to adapt to the changing environment of their host. Pathogenic bacteria (e.g. Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae and Bordetella pertussis) contain simple-sequence repeats in coding regions or promoters of loci that encode virulence factors. These sequences are termed ‘contingency loci’ (28). The mechanism of mutation of these hypervariable loci is also thought to result from slipped strand mutagenesis. Analysis of the N.meningitidis genome for repetitive sequences in known and strong candidate contingency loci revealed that 28/42 (67%) loci contained G- or C-runs (29). Similarly, B.pertussis contains a hypervariable C15 run in the promoter region of the fim gene (30) and gonococcal bacteria have a C12 run in the promoter of the FetA gene (31). Although the majority of mononucleotide repeats in contingency loci contain G- or C-runs, non-pathogenic E.coli have very few microsatellite sequences, and slippage is limited to non-coding runs of mononucleotides (32); moreover, in the (non-pathogenic) E.coli K12 genome, there is a low incidence of G- or C-runs relative to A- or T-runs, and there is a bias against the use of GGG and CCC codons (24). It is, therefore, tempting to suggest that the hyperinstability of G-runs has allowed for selection in favor of these sequences during evolution of the genomes of pathogenic bacteria and against their use in other organisms.
In humans, mononucleotide repeats are of particular interest because a number of cancer-related genes contain mononucleotide repeat sequences in coding regions, which have been shown to be prone to mutations in microsatellite-unstable tumors. These genes usually contain units of 8–10 mononucleotide repeats in which frameshifts give rise to inactivation of the protein. Most of these genes, including TGFBR2 (33), hMSH3 (34), TCF4 (35), casp5 (36), RAD50 (37), BLM and APC (38), contain A-runs; a few contain G- or C-runs [Bax (39), hMSH6 (34) and IGF2R (39)]. The bias toward runs of A over runs of G in these sequences may be generally protective against inactivation, based on the relative mutation rates of the mononucleotides. The frequencies with which mutations are observed in these genes must also be related to the selective advantage that the inactivation of the corresponding protein provides to the tumor cells. The gene that is most frequently observed to undergo such frameshifts in MMR– colorectal cancers is TGFBR2, in which the target is a run of As (40,41).
It is interesting that the mutation rates of (CA)17 and A17 repeats were similar in MMR+ cells. It might have been expected that mononucleotide runs would generally be less stable than dinucleotide runs, partly based on our previous observation that a tetranucleotide repeat tract is much more stable than the CA-dinucleotide tract (42). Our results on mononucleotide runs show clearly that the exact base composition of the repeat can have a strong influence on the degree of instability. We are currently studying other dinucleotide repeats, which should contribute to our understanding of how base composition of the repeat can affect the relative instabilities of mono- and dinucleotide repeats.
In the experiments presented here, where the target mononucleotide runs are inserted to produce a selectable reporter sequence in the –1 reading frame, we found mixed results on the types of mutations in A-runs versus G-runs in MMR+ and MMR– cells (Table 2). Both types of MMR+ cells exhibited a strong propensity for +1 mutations in G-runs. This bias was also found for A-runs in the MMR+ CAK cells. (No mutants were recovered from hTERT 1604 cells containing the A-run.) In the MMR– cells, however, the A-run produced mostly –2 frameshifts and the G-run produced an equal proportion of +1 and –2 mutations. In a similar experiment using a (CA)17 repeat in both reading frames, we previously found an excess of insertions over deletions in both MMR+ and MMR– cells (18). Dinucleotide repeats may have a different pattern of frameshift mutations from that of mononucleotide repeats. Using forward mutation systems, other laboratories have reported similar biases. Sia et al. (7), using a G18 repeat target, reported a preference for insertions in wild-type yeast, but mostly deletions in msh3 and msh6 strains. Similarly, Tobi et al. (43) reported that in a tRNA-based shuttle vector system containing G-runs, the hmsh6-deficient MT1 human lymphoblastoid line showed a preference for single base deletions, whereas MMR+ cells (TK6) did not. In another forward mutation system in yeast, Tran et al. (44) reported a strong bias for deletions in A-runs in both wild-type and msh2 strains. This result is consistent with our data in MMR– cells. Since the most common frameshift mutations in repetitive sequences tend to involve a single repeat, it would be necessary to extend our studies, analyzing the microsatellites in the +1 reading frame, in order to obtain an unbiased comparison of mononucleotide insertions versus deletions. Our data would then be more directly related to those of the forward mutation assays, in that insertions and deletions of the same size could be compared directly, as in our study of CA repeats (18).
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MATERIALS AND METHODS |
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Cell culture
CAKStu3 is a thymidine-kinase deficient derivative of CAK, an immortalized, non-tumorigenic line of embryonic mouse fibroblasts with a near-diploid chromosome complement (45). This cell line is mismatch repair proficient (13). These cells were cultured in Eagle’s minimal essential medium with 10% iron-supplemented calf serum (Hyclone, Logan, UT).
H6 is a subclone of the human colorectal cancer cell line HCT116 (11), which lacks mismatch repair activity as the result of the absence of a normal copy of the hMLH1 gene (12). It was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% iron-supplemented calf serum.
hTERT1604 is a normal human lung fibroblast cell line that was immortalized by the introduction of hTERT, the catalytic subunit of the human telomerase gene (46). These cells were obtained from Dr Roger Schultz (University of Texas Southwestern Medical center at Dallas). They were grown in DMEM supplemented with 10% fetal bovine serum (Sigma, St Louis, MO) and twice the normal concentration of non-essential amino acids (Life Technologies, Rockville, MD).
All cells were maintained without antibiotics at 37°C in 5% atmospheric CO2.
Plasmid construction
Plasmids containing various microsatellite sequences were constructed by insertion of oligonucleotides containing the repetitive sequences into the parent plasmid, pCon (Fig. 1). pCon contains a fusion gene comprised of the herpes simplex virus thymidine kinase gene (tk) fused at its 3' end to the 5' end of a bacterial gene coding for neomycin resistance (neo). The oligonucleotides were inserted at an AatII site near the 3' end of the tk gene, such that the neo gene is in the (–1) reading frame. Cells with mutations in the repeat sequences that restore the reading frame are resistant to the neomycin analog G418. The bacterial hygromycin-resistance gene (hyg) was used for the selection of stable transfectants.
pCon was derived from the pRTM2 plasmid (13) and was designed so that derivatives containing different microsatellites could be constructed in a single step (Fig. 1A). Site-directed mutagenesis (QuickChange Site-directed mutagenesis kit, Stratagene, La Jolla, CA) was used to remove an AatII site in the hyg gene and to add an AatII site to the 3' end of the insert containing the CA-repeat. The original CA repeat and its surrounding sequences were removed by digestion with the restriction enzyme AatII and the plasmid was re-ligated. All microsatellite-containing plasmids, except the CA-repeat plasmid pRTM2, were made by insertion of repetitive sequences, flanked on each side by 5 bp of unique sequence, into the unique AatII site of pCon. One of the G17 plasmids (pG17) was made by the inverted insertion of an oligonucleotide containing a C17-repeat; therefore, the sequences flanking the microsatellite were different from the other plasmids. The pRG17 plasmid has microsatellite-flanking sequences that are identical to those of the other plasmids. This plasmid was constructed in order to rule out possible effects of the different flanking sequences on mutation rates. Sequences of the oligonucleotide inserts are shown in Figure 1B.
Transfection and fluctuation analysis
Plasmid DNA (1 µg) was linearized with HindIII and introduced into 107 cells by electroporation. Transformed clones were selected with hygromycin B (150 µg/ml for all lines except hTERT1604, which required 200 µg/ml). Independent hygromycin-resistant (hygR) clones were isolated from different plates and the cultures were expanded. Multiple subcultures were established from each hygR clone (100 cells per subculture for CAK and hTERT1604 cells and five cells per subculture for H6 cells). Ten subcultures for each hygR clone assayed were expanded to 1–5 x 106 cells each. Mutants that expressed the neo gene were selected by plating cells in medium containing G418 (500 µg/ml; Geneticin, Life Technologies). Cells were plated at the following densities: CAK, 2–5 x 105 cells per 100 mm dish; hTERT1604, 1–5 x 105 cells per dish; and H6, 0.02–2 x 105 cells per dish. (These densities were determined on the basis of preliminary analyses of mutation frequencies of each transfected clone.) One G418R colony was isolated from each plate and expanded for DNA isolation and PCR analysis. Plates were fixed and stained with a solution of 0.2% crystal violet in 50% methanol or fixed in 100% methanol and stained with a 1:50 dilution of Giemsa stain in phosphate-buffered saline. The number of G418R colonies was determined and was adjusted for the colony-forming efficiency of each subculture in non-selective media. Mutation rates were calculated using the Chipmunk BASIC computer program (written by Eric Bronner, Oregon Health Sciences University, Portland, OR). The fluctuation analysis program is based on the Luria and Delbrück (10) method of the mean, using the Capizzi and Jameson (47) tables.
The transfection conditions were designed to result in clones containing one microsatellite insert. Using the above transfection conditions, efficiencies average 2 x 10–5 per cell (data not shown) with most clones containing one insert. Clones that have multiple inserts can be identified by the PCR product profiles of the DNA from the G418R clones. Clones with more than one insert exhibit a normal-sized PCR product in addition to the PCR product with the frameshift mutation. The number of inserts can be determined by PhosphorImager analysis of PCR products that are separated on 6% polyacrylamide gels. All of the transformants presented here contained single inserts.
Since the location of the insertion of the transfected microsatellite DNA into the genome is a non-targeted event, we analyzed at least three independent hygR clones for each microsatellite sequence. This analysis of several clones was done to control for variation among mutation rates that might have resulted from differences in sequence context at the integration sites.
Statistical analysis
Statistical analysis of the difference in mutation rates between A17 and G17 in H6 cells was carried out as described previously (18). Rates for each subculture were determined by the method of the median (48). The median of all rates to be compared was determined, and the numbers of subcultures of each type whose rates fell above or below the mean were tabulated. A Fisher’s exact test was carried out on these numbers.
PCR
DNA was isolated from hygR and G418R clones using the DNAeasy tissue kit by Qiagen (Valencia, CA). PCR was performed using either radioactively labeled or fluorescently labeled primers. Primer sequences were 5'-CAACGGCGACCTGTATAACG-3' and 5'-GATTGGTCGTAATCCAGGAT-3' (13).
For radioactive PCR reactions, the 5'-primer was end-labeled with [
-33P]ATP and polynucleotide kinase (New England Biolabs, Beverly, MA). A 10 µl PCR reaction mixture contained 1x manufacturer-supplied reaction buffer, 200 µM each dNTP, 1.25 µM each primer, 0.125 U Taq DNA polymerase (Life Technologies) and 100–500 ng genomic DNA. For fluorescent PCR reactions, the 3'-primer was end-labeled with either HEX or FAM. The fluorescently labeled primers were purchased from Synthegen (Houston, TX). The reaction mixture was similar to that described above, except that a 20 µl reaction volume was used and the primer concentrations were 0.625 µM each. Amplification conditions were as follows: initial 10 min denaturation at 94°C; 30 cycles each of 94°C for 30 s; 60°C for 30 s and 72°C for 30 s; and final 10 min extension at 72°C.
33P-labeled PCR products were separated on 6–10% denaturing polyacrylamide gels (1700 V; 1.5–4 h). Gels were dried and exposed to Kodak BioMax film at room temperature. Fluorescently labeled PCR products were analyzed using capillary electrophoresis (ABI Genetic Analyzer 310, PE Biosystems, Foster City, CA). In some analyses, HEX-labeled PCR products from G418R DNAs were compared to FAM-labeled PCR products from control DNA by subjecting both PCR products to electrophoresis in the same sample. In other analyses, the PCR products from both control and mutant DNAs were labeled with HEX-primers and run separately on the 310 Genetic Analyzer. Using internal TAMRA-labeled size standards (GeneScan-500) the electrophoresis profiles from the control and mutant DNAs were directly compared. The samples were run using either POP6 polymer with 2.0 kV injection voltage, 30 s injection time, 15 kV run voltage, 50°C run temperature and a run time of 29 min or POP4 polymer with 15 kV injection voltage, 1–5 s injection time, 15 kV run voltage, 60°C run temperature and a run time of 24 min. The data were processed using ABI 310 software (GeneScan 3.1.2, using filter set A).
Determination of mismatch repair efficiencies
Repair efficiencies were calculated using the following equation: EC = [(RM – RWT)RM] x 100, where EC = the percentage efficiency of correction, R = mutation rate, M = mutant and WT = wild-type (7). The rate for a particular type of mutation was determined by multiplication of the frequency of that type of mutation by the overall mutation rate.
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ACKNOWLEDGEMENTS |
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We thank E.Bronner and Dr R.M.Liskay for the Chipmunk program and Drs W.K.Kaufmann and T.D.Petes for helpful comments on the manuscript. This work was supported by National Institutes of Health grant CA63264. K.R. was supported by National Science Foundation grant DBI-9605149, Research Experiences for Undergraduates. N.A.Y. is a Howard Hughes Medical Institute Predoctoral Fellow.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 919 966 6921; Fax: +1 919 966 0717; Email: jayne_boyer@med.unc.edu
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REFERENCES |
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