Mutagenesis Advance Access originally published online on January 4, 2008
Mutagenesis 2008 23(2):87-91; doi:10.1093/mutage/gem047
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The frequency of CC to TT tandem mutations in mismatch repair-deficient cells is increased in a cytosine run
1Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA 2Center for Research on Occupational and Environmental Toxicology 3Department of Molecular and Medical Genetics, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA
Mononucleotide runs are hot spots for frameshift mutations in mismatch repair (MMR)-deficient cells. However, a role for mononucleotide runs in the formation of base pair substitutions has not been tested. Previously, we demonstrated that ultraviolet radiation C (UVC)- or reactive oxygen species-induced CC to TT tandem mutations are markedly enhanced in MMR-deficient cells. The target for the mutational analysis was two cytosines in a run of five cytosines (5C) within mouse Aprt. Because mutation from C to T for either or both of the two critical cytosines created a codon yielding a functional Aprt protein, this assay allowed both single and tandem substitutions to be quantified and the relative ratios compared. To determine if the cytosine run increased the frequency of single and/or tandem base pair substitutions, alternative constructs were created in which the cytosine run was disrupted by flanking the target cytosines with either thymines (2Cpyr) or adenines (2Cpur). Disruption of the cytosine run dramatically decreased the frequency of UVC-induced tandem mutations in the 2Cpyr and 2Cpur constructs, as compared with the 5C construct. Moreover, CC to TT tandem mutations occurred spontaneously or were induced by oxidative stress only within the 5C construct. These results demonstrate that CC to TT tandem mutations in MMR-deficient cells form more readily in a homocytosine run than in a sequence limited to two cytosines.
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Introduction |
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The CC to TT tandem substitution is a signature for ultraviolet (UV) mutagenesis because it is observed commonly in the P53 and PTCH genes in sun-exposed skin cancers (1
,2). Even higher frequencies of CC to TT mutations have been observed in skin cancers from persons deficient for nucleotide excision repair (3) or translesion polymerase pol 
(4) (i.e. xeroderma pigmentosum patients), thus demonstrating a role for these pathways in preventing formation of the tandem mutation after UV exposure.
In prior work, we demonstrated that mismatch repair (MMR) also plays a role in preventing UV or oxidative stress-induced tandem mutations (5). The primary function of MMR is to maintain genetic stability by repairing mismatched bases and small insertions/deletions caused by spontaneous or damage-related DNA polymerase errors (6–8). Most studies examining the role of MMR in preventing mutations have focused on errors caused by DNA polymerase slippage in mononucleotide runs leading to formation of frameshift mutations. These studies have demonstrated a role for MMR in maintaining homonucleotide run stability in sequences containing as little as four consecutive homonucleotides (9). For example, a run of six guanines within mouse Hprt is a hot spot for frameshift mutation frequencies in Pms2-deficient mice (10). Other examples of frameshift mutations within mononucleotide runs in MMR-deficient cells have been reported (9,11–15).
The focus on frameshift mutations within mononucleotide runs in MMR-deficient cells has left unanswered the question of whether these runs also increase or alter the formation of base pair substitutions. Our study of spontaneous and induced mutations in MMR-null cells focused on two cytosines within a run of five cytosines in a mutated version of mouse Aprt (5). Mutation of the target cytosines to thymine, either singly or in tandem, was required for restoration of gene function. Here, we extend the prior study to show that disruption of the cytosine run dramatically decreases formation of tandem CC to TT mutations at the target codon in Pms2-null cells.
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Materials and Methods |
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Generation of reversion constructs
The three Aprt reversion constructs used for this study (see Figure 1) were created with site-directed mutagenesis using methods described elsewhere (16
). These reversion constructs were linked to the bacterial puromycin resistance gene (pur) and transfected into a Pms2, Aprt-null mouse kidney cell line using methods previously described (16). Transfectants containing one copy of integrated plasmid, as verified by Southern blot analysis, were used for this study. Two independent cell lines for each construct were tested.
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Genotoxin treatments
Prior to genotoxin exposures, the cells were grown in 4 µg/ml 2-fluoroadenine (Sigma, St Louis, MO, USA) to eliminate preexisting Aprt revertants. For ultraviolet radiation C (UVC) exposure, one million cells were seeded overnight in a 100-mm dish. The UV lamp (Spectronic Corporation, Westbury, NY, USA) output was measured with an International Light Meter (International Light Technologies, Newburyport, MA, USA) prior to cell irradiation. For irradiation, the cells were rinsed with phosphate-buffered saline, the liquid was removed and the cells exposed to 7–9 J/m2. After irradiation, the cells from each dish were trypsinized and allowed to recover for 5–7 days in T75 flasks. For other cell exposures, 8 x 105 cells were seeded overnight in T75 flasks. The cells were then treated with the alkylating agent ethyl methanesulfonate (EMS) (400 or 600 µg/ml) or the Fenton reaction (15 µM hydrogen peroxide plus 150 µM each CuSO4 and FeSO4) for 24 h. Following genotoxin exposures, the cells were allowed to recover for 5–7 days. Untreated cells were handled similarly to treated cells to provide spontaneous revertant frequency data.
Reversion assay
After the recovery time, 2–6 million cells were plated into 125 x 25-mm dishes (BD Falcon, San Jose, CA, USA) at a density of 1.5–2.0 million cells per dish. Twenty-four hours after seeding, the cells were exposed to medium containing 58 µM azaserine (Sigma) and 76 µM adenine (Sigma) (AzA medium) to select for Aprt revertants (16). Only cells expressing a functional Aprt gene can grow in AzA medium. Aprt revertant clones were isolated 2–3 weeks later and expanded until confluent in T25 flasks, at which time DNA was isolated and prepared for sequencing. Parallel dishes were stained with crystal violet, as were cloning efficiency dishes, to determine revertant frequencies.
Sequence analysis
Individual clones were isolated and expanded for DNA isolation. PCR was performed to amplify exon 3 of Aprt as described elsewhere (17). Sequencing was performed at the Molecular Microbiology and Immunology Research core facility at Oregon Health & Science University. To ensure that one mutational event did not account for multiple samples, no more than three revertant clones were examined per independent ‘pot’.
Statistical analysis
Data were analyzed using two statistical approaches: the non-parametric Kruskal–Wallis test and a quasi-likelihood approach (to accommodate revertant frequencies that were near zero). Both approaches yielded similar conclusions.
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Results |
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Spontaneous revertant frequencies and mutational spectra
The target site for these experiments was derived from site-directed mutagenesis of two thymines in codon 69 of mouse Aprt (Figure 1A) to create two cytosines (Figure 1B). Mutation of either cytosine or of both (i.e. a tandem mutation) to thymine (Figure 1C) creates a codon that produces a functional Aprt protein, which can be selected with the reversion assay. The target cytosines are within a run of five cytosines for the 5C construct, whereas they are flanked by thymines in the 2Cpyr construct and adenines in the 2Cpur construct (Figure 1B). Data obtained with the 5C construct have been reported previously (5
). The average spontaneous revertant frequency for Pms2-null cells containing the 5C construct was 37.2 x 10–6 (Table I). When the target cytosines were flanked by thymines in the 2Cpyr construct, the average revertant frequency decreased to 4.2 x 10–6 (Table I). Similarly, when the target cytosines were flanked by adenines in the 2Cpur construct, the average spontaneous revertant frequency was 4.8 x 10–6. The spontaneous revertant frequencies for the 2Cpyr and 2Cpur constructs were different that for the 5C construct (P = 0.001), which demonstrates that a run of five cytosines predisposes MMR-deficient cells to a higher frequency of spontaneous revertants.
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A sequence analysis for eight spontaneous revertant clones bearing the 2Cpur construct demonstrated single C to T base pair substitutions, six at the 5' cytosine position and two at the 3' cytosine (Table II). Unexpectedly, none of the spontaneous 2Cpyr revertants that were isolated and expanded for sequence analysis survived the expansion process and therefore data are not available to describe the spontaneous spectrum of mutations for this construct. Most of the spontaneous revertant cells bearing the 5C construct exhibited single C to T substitutions (88%), with the remainder (12%) being CC to TT tandem substitutions.
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Revertant frequencies and mutational spectra in UVC-exposed MMR-deficient cells
The average revertant frequency observed in the UVC-exposed cells containing the 5C construct was 53.4 x 10–6. UVC-induced revertant frequencies for cells containing the 2Cpyr and 2Cpur constructs were lower at average frequencies of 21.7 x 10–6 and 14.2 x 10–6, respectively. These differences were marginally significant (P = 0.09 and 0.07 for the 2Cpyr and 2Cpur constructs, respectively).
The sequence analysis confirmed a UV mutagenic effect for all three constructs. For the 2Cpur construct, one tandem CC to TT mutation (4%) was induced. The remainder (96%) was single C to T mutations. Interestingly, while most spontaneous C to T mutations for this construct were at the 5' cytosine of the target sequence (75%), most (84%) were observed at the 3' cytosine after UVC exposure (P = 0.002).
Unlike spontaneous revertants for cells bearing the 2Cpyr construct, all revertant clones isolated after UVC exposure grew well and yielded DNA preparations for a sequence analysis (Table II). Three tandem CC to TT mutations (9% of total) were observed at the target site. One additional tandem substitution was observed for the 2Cpyr construct, a CT to TA mutation, which was apparently due to mutation of a CT photodimer in the TCCT sequence. In addition, a complex mutation with three non-adjacent base pair substitutions was observed in the 2Cpyr construct (TCCTC to CCTTT). The remaining mutations (84%) were single C to T substitutions.
The relative increase in revertant frequencies after UVC exposure for cells bearing the 5C construct was modest (37.2 x 10–6 in unexposed cells; 53.4 x 10–6 in exposed cells), but a significant shift in spectrum was observed because the percentage of CC to TT tandem mutations rose from 12 to 64% (P < 0.001) (Table II). All but two (i.e. 14 of 16) of the CC to TT substitutions observed for the 5C construct occurred within the target codon (i.e. 56% of total mutations were tandem CC to TT substitutions in the target sequence) (Table II). The remaining two CC to TT tandem mutations included the 5' cytosine in the target sequence and the upstream adjacent cytosine (CCCCC to TTCCC).
Multiplication of revertant frequencies by the relative percentages of single or tandem mutations (at the target site only) to obtain approximate frequencies for these mutations revealed little difference for the formation of single C to T mutations in UVC-exposed cells (Table III). However, a dramatic difference was observed for formation of the CC to TT tandem mutation, which occurred 15- and 50-fold more frequently for the 5C construct than for the 2Cpyr and 2Cpur constructs, respectively (Table III).
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Revertant frequencies and mutational spectra in cells exposed to reactive oxygen species
The average revertant frequency for cells containing the 5C construct exposed to a mixture of hydrogen peroxide and metals to generate reactive oxygen species (ROS) was comparable to that for UV exposure (Table I). Moreover, very high-frequency formation of CC to TT mutations at the target site was observed (94% of total mutations) (Table II). In contrast, the revertant frequencies after ROS exposure for cells containing the 2Cpyr and 2Cpur constructs were at or below the background levels (Table I) (P = 0.02 for both comparisons), and few revertants were recovered. All revertants that were recovered yielded single C to T substitutions at the 3' cytosine (Table II).
Revertant frequencies following exposure to EMS
UVC induces C to T mutations at the target codon. To determine if the guanine run on the opposite strand was also susceptible to induced mutations, we tested the alkylating agent EMS because it induces G to A mutations. The average revertant frequencies for cells containing the 2Cpyr and 2Cpur constructs were comparable at 95.0 x 10–6 and 70.7 x 10–6, respectively. The average EMS-induced revertant frequency for cells with the 5C construct was higher at 428 x 10–6 (Table I), but due to variation between experiments, the differences between the 5C construct and the other constructs were not statistically different (P = 0.1 for both comparisons). All EMS mutations detected for all constructs were single G to A substitutions (Table II).
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Discussion |
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A role for MMR in preventing CC to TT tandem mutations was shown in a prior study in which we compared mutations in the 5C construct in DNA repair-proficient cells with those in Pms2-null cells (5
). The current follow-up study examines whether CC to TT tandem mutations occur more frequently in MMR-deficient cells when the target site is within a run of cytosines. The results show that tandem mutations form readily within the cytosine run, but form infrequently when the cytosine run is disrupted. Our study, therefore, provides evidence that a mononucleotide run can increase the frequency of a non-frameshift mutation in MMR-deficient cells, in this case the CC to TT tandem mutation. Though well studied (18–21), the underlying mechanism for formation of the CC to TT tandem mutation in intact genomes has not been elucidated.
Spontaneous CC to TT mutations were observed in 12% of revertant cells carrying the 5C construct. The percentage of tandem mutations rose to 94% after these cells were exposed to a mixture of metals and hydrogen peroxide. Neither spontaneous nor ROS-induced CC to TT mutations were observed for the 2Cpyr and 2Cpur constructs. Although these observations demonstrate that the cytosine run is inherently more prone to the CC to TT mutation, they are difficult to interpret because the initiating events for the spontaneous and ROS-induced tandem mutations are unknown. In contrast, the origin of UVC-induced CC to TT mutations is the CC photodimer (22), which can be formed at the target site for all three constructs. The observation that 56% of mutations for the 5C construct in UVC-exposed cells were tandem CC to TT substitutions at the target codon, as compared with only 9 and 3% tandems for the 2Cpyr and 2Cpur constructs, respectively, demonstrates a sequence effect from the homocytosine run. Moreover, when revertant frequencies were also considered, the absolute induction of CC to TT mutations was found to be 15- to 50-fold higher when the cytosine run was present (see Table III). Assuming that cytosine dimers at the target site occurred at the same relative frequencies in the three constructs, the results demonstrate that sequence context influences the relative probabilities that single C to T mutations or tandem CC to TT mutations form after UV exposure.
The possibility that the type of mutation that is formed (i.e. single versus tandem mutation) is not strictly a function of the initiating UV damage is also supported by a recently completed study in which we showed that pretreatment of DNA repair-proficient cells with oxidative stress dramatically increased UV induction of tandem mutations for the 5C construct. In that study, the percentage of UVB-induced CC to TT tandem mutation rose from 2% without oxidative stress to 40% in cells maintained under oxidative stress for 24 h; however, UVB-induced revertant frequencies were not altered by the presence of oxidative stress (21). Thus, in those experiments, processing of the cytosine dimer appears to have been altered in such a way as to produce more tandem mutations when the cells were under oxidative stress.
Replication past UV damage requires a translesion polymerase. Of these, pol is most capable of high accuracy bypass of CC photoproducts (23). The accuracy of pol when copying past a cytosine dimer within a cytosine run, however, has not been tested. Moreover, other translesion polymerases such as pol 
(24) and pol 
(25) can also bypass UV damage, but with lower accuracy. For example, the frequency of UVC-induced CC to TT tandem mutations increases >50-fold in pol -deficient yeast (24). We suggest that the higher induction of CC to TT tandem mutations by UVC in the 5C construct is due to decreased pol accuracy within a cytosine run when performing dimer bypass and/or the increased use of less accurate translesion polymerases. If this hypothesis was correct, the role for MMR in preventing the tandem mutations would be to correct misinsertions that occur after mutagenic bypass. Such a role would be similar to the MMR function of correcting insertion or deletion loops that occur when a replication or translesion polymerase copies a mononucleotide run (6–8,26).
Finally, we used EMS mutagenesis to examine the target sequence of two guanines on the opposite strand. The results showed similar mutagenic responses for cells bearing the 2Cpyr and 2Cpur constructs, that both constructs could respond well to an alkylating mutagen, and suggested a role for the cytosine run in the mutagenic response. Only single G to A mutations were induced in the guanine run, as opposed to tandem mutations within the cytosine run, for all three constructs.
In summary, we used a reversion assay to examine mutagenesis at a two base pair target in mouse Aprt in different sequence contexts in MMR-null cells. The induction of CC to TT tandem mutations by UVC was dramatically lower when the target cytosines were flanked by thymines or adenines, as opposed to being included with a cytosine run. This result, combined with the observations of elevated spontaneous and oxidative stress-induced CC to TT mutations in the cytosine run, demonstrates that the presence of a cytosine run can promote formation of the distinctive CC to TT tandem mutation.
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Funding |
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National Institutes of Health (CA 76528 to M.S.T., T32 ES07060 and P42 ES10338 to A.M.S).
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Acknowledgments |
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We thank Drs Amanda McCullough and Andrew Buermeyer for helpful discussions, Dr John Hays for suggesting these experiments and Mike Lasarev for statistical help. A.M.S. received assistance for supplies as contributing editor-assistant for Science Aging Knowledge.
Conflict of interest statement: None declared.
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Notes |
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* To whom correspondence should be addressed. Tel: +1 503 494 2168; Fax: +1 503 494 3849; Email: turkerm{at}ohsu.edu
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Received on September 28, 2007; accepted on November 17, 2007.
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