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Molecular and Cellular Biology, January 2004, p. 465-474, Vol. 24, No. 1
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.1.465-474.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Monica Francesca Blasi,1, Federica Chiera,1 Paola Fortini,1 Paolo Degan,2 Peter Macpherson,3 Masato Furuichi,4 Yusaku Nakabeppu,4 Peter Karran,3 Gabriele Aquilina,1 and Margherita Bignami1*
Chemical Carcinogenesis Unit, Istituto Superiore di Sanità, Rome,1 Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy,2 Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms, United Kingdom,3 Department of Biochemistry, Kyushu University, Fukuoka, Japan4
Received 6 August 2003/ Returned for modification 10 September 2003/ Accepted 18 September 2003
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ABSTRACT |
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INTRODUCTION |
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and hMutSß mismatch recognition factors are heterodimers of hMSH2/hMSH6 and hMSH2/hMSH3, respectively. hMutS preferentially initiates correction of base-base mismatches and small IDLs, whereas hMutSß targets larger IDLs (for reviews, see references 26 and 31). Complete excision and replacement of the mismatched section of DNA also involves heterodimeric complexes between the hMLH1 and hPMS2 (or hMLH3) proteins, PCNA (43), RPA, DNA polymerase 
, and hEXO1 (44). Because of its central role in replication error correction, cells in which MMR is incapacitated by inactivating mutations in hMSH2, hMLH1, hPMS2, or hMSH6 have high spontaneous mutation rates. This mutator effect is observed as a dramatic increase in the frequency of base substitutions and frameshifts. Frameshifts derive from uncorrected IDLs and are generally located in repetitive DNA sequences. These are located within the coding sequences of genes, as well as in the numerous noncoding microsatellite regions distributed throughout the genome. Alterations in the length of multiple microsatellites are a characteristic feature of MMR-deficient cells, and this microsatellite instability (MSI) is diagnostic for MMR deficiency in both cell lines and tumors (1).
MSI is a defining feature of tumors arising in hereditary nonpolyposis colorectal cancer (HNPCC) families. HNPCC individuals have a germ line mutation in one of the MMR genes—most commonly hMSH2 or hMLH1. The increased mutation rate that accompanies somatic inactivation of the second allele accelerates the development of colorectal and other typical cancers in these individuals. In addition to these familial cases, a significant proportion of sporadic cancers exhibit MSI. In these cases, epigenetic silencing of an MMR gene, most commonly hMLH1, inactivates MMR. Organs, tumors, and cell lines from MMR gene knockout mice recapitulate this genetic instability and display both increased mutation rates and MSI (for a review, see reference 11). By analogy to HNPCC, the cancer proneness of these mice is generally considered to reflect an increased rate of accumulation of inactivating mutations in key target genes that normally function to prevent unlimited cellular proliferation.
Although it has been generally assumed that the mutator phenotype of MMR-deficient cells reflects uncorrected spontaneous DNA polymerase errors (42), MMR is also known to process some altered or damaged DNA bases. For example, O6-methylguanine, a lesion induced by treatment with methylating carcinogens, is known to miscode during DNA replication and DNA containing O6-methylguanine base pairs is bound by hMutS (10, 17, 30). As a result, MMR-defective cells are hypermutable by these agents (3) and this hypermutability by spontaneous DNA lesions is a potential contributor to the mutator phenotype.
Oxidation is a significant and constant source of spontaneous DNA damage. The oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG) is a particularly frequent DNA lesion that, during replication, can form base pairs with adenine (41) to promote the formation of G 
T transversions (46, 12). The extent of the threat posed by DNA 8-oxoG is emphasized by the existence of a highly conserved three-tier system that protects against the mutagenic properties of 8-oxoG. Two complementary arms of the base excision repair (BER) pathway bring about the removal of 8-oxoG from DNA. In the first, the hOGG1 DNA glycosylase initiates excision of the oxidized purine from resting DNA in which 8-oxoG is paired with C. An additional BER pathway, initiated by the MYH DNA glycosylase, a homolog of the Escherichia coli MutY protein, removes adenine misincorporated opposite 8-oxoG during replication. This promotes the eventual removal of the oxidized purine from DNA via hOGG1-mediated processing of the 8-oxoG · C base pairs generated during repair. Purine dNTPs are also subject to oxidative damage, and the oxidized products are substrates for incorporation into DNA during replication. To avoid this, human cells sanitize the dNTP pool by hydrolyzing oxidized purine dNTPs. This degradation is carried out by hMTH1, a homolog of the E. coli MutT protein that hydrolyzes 8-oxodGTP (36).
There is mounting evidence that MMR also contributes to reducing the burden of oxidized DNA bases. Treatment of E. coli MMR mutants with H2O2 increases the instability of repetitive sequences in extrachromosomal plasmid DNA (25). In Saccharomyces cerevisiae, the mutator phenotype of MSH2- and MSH6-defective strains is significantly decreased by anaerobic growth, suggesting that a large fraction of spontaneous mutagenesis in MMR-deficient strains is caused by the persistence of oxidatively damaged bases (18). Yeast MMR processes 8-oxoG · A base pairs, and its ability to excise A incorporated opposite 8-oxoG may compensate for the apparent absence of a MutY homolog in this organism (37). The mammalian MMR system also participates in minimizing the levels of oxidative DNA damage. Thus, the steady-state level of DNA 8-oxoG is significantly elevated in MMR-deficient mouse (15) and human (14) cells. Consistent with this, hMutS binds to some 8-oxoG-containing base pairs (35). The efficiency of excision of the oxidized purine by BER appears to be similar in MMR-proficient and -defective cells. We have provided evidence that MMR removes 8-oxodGMP incorporated during replication (14) and suggested that this new role for MMR represents a fourth level of protection against the dangers of oxidized DNA bases.
To determine the full impact of incorporated oxidized DNA precursors on spontaneous mutation in MMR-defective cells, we characterized spontaneous mutations in msh2-/- mouse embryo fibroblasts (MEFs) in which hMTH1 cDNA is expressed. We also analyzed the influence of increased hMTH1 expression on MSI. The findings indicate that (i) the oxidized purine dNTP pool is a significant contributor to mutation in MMR-deficient cells, (ii) both 8-oxodGTP and 2-oxodATP are implicated in mutation, and (iii) incorporation of oxidized DNA precursors is a significant influence on MSI in repair-defective human tumor cells.
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MATERIALS AND METHODS |
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-32P]dGTP (2 Ci/mmol; Amersham), and cell extract. Following 10 min of incubation at 37°C, reactions were terminated by chilling to 0°C and addition of EDTA to 10 mM. Aliquots (2.5 µl) were applied to thin-layer polyethyleneimine-cellulose plates, which were developed in 1 M LiCl. Dried plates were exposed to X-rays film, and dGTP and dGMP were quantitated by using the National Institutes of Health V1.59 software package. One unit of hMTH1 produces 1 pmol of dGMP per min in the standard assay. For Western blotting cell extracts were separated by sodium dodecyl sulfate-7.5% polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane with a Trans-Blot cell apparatus (Bio-Rad), and probed overnight with anti-hMTH1 antibody, followed by the appropriate secondary antibody. Blots were developed by using the ECL detection reagents (Amersham).
8-OxoG determinations.
8-OxodG was measured by high-performance liquid chromatography with electrochemical detection (HPLC/EC) as previously described (7) following DNA extraction, RNase treatment, and enzymatic hydrolysis. DNA was extracted by a high-salt protein precipitation method. Briefly, cells were lysed with sodium dodecyl sulfate and digested with protease (Qiagen) at 37°C for 1 h. Proteins were precipitated by adding NaCl to 1.5 M, and DNA in the supernatant was collected by addition of 2 volumes of ethanol. The DNA pellet was resuspended in Tris-EDTA, incubated with RNases A and T1 at 37°C for 1 h, and precipitated again with ethanol. Enzymatic digestion was then performed at 37°C with nuclease P1 (Boehringer Mannheim) for 2 h and alkaline phosphatase (Boehringer Mannheim) for 1 h. Enzymes were precipitated by addition of CHCl3, and the upper layer was stored for analysis of 8-oxodG at -80°C under N2. The DNA hydrolysate was analyzed by HPLC/EC (Coulochem I; ESA Inc.) with a C18 5-µm Uptishere column (250 by 46 mm; Interchim) equipped with a C18 guard column. The eluent was 50 mM ammonium acetate, pH 5.5, containing 9% methanol, at a flow rate of 0.7 ml/min. The potentials applied were 150 and 400 mV for E1 and E2, respectively. The retention time of 8-oxodG was 
23 min. Deoxyguanosine was measured in the same run of corresponding 8-oxodG with a UV detector (model SPD-2A; Shimadzu) at 256 nm; the retention time was 17 min.
Mutation rate analysis at the hprt gene and DNA sequencing of mutants. Cells were plated at low density (100 cells/dish) and grown in complete medium to a density of 0.4 x 106 to 1 x 106 per dish before plating of the entire culture (50 to 60 independent cultures) into medium supplemented with 6-thioguanine (5 µg/ml; Sigma). The mutation rate was calculated as µ = M C-1 ln2, where C is the number of cells at selection time and M is -ln(P0), where P0 is the proportion of cultures with no mutants. One 6-thioguanine-resistant mutant was isolated per culture to ensure mutation independence. Cytoplasmic RNA was extracted and used to synthesize hprt cDNA by SuperScript One-Step reverse transcription-PCR (Invitrogen) in a final volume of 50 µl containing 1 µg of RNA, 0.2 µM primers (45), RT/Platinum TaqMix (1 µl), and buffer provided with the enzyme. Cycles included 30 s at 55°C; 2 min at 94°C; 35 cycles of 15 s at 94°C, 30 s at 55°C, and 1 min at 72°C; and a final 10 min at 72°C. Reverse transcription-PCR products were cleaned with a QIAquick PCR purification kit (Qiagen), used in sequencing reactions (25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C), and analyzed with an ABI Prism 310 automatic sequencer.
MSI. Genomic DNA was isolated from subclones of clonal isolates of DLD1, DLD1/clone 2A, DU145, and DU145/clone 1. Ninety-six-well plates were seeded at a density of <1 cell/well, and DNA was prepared from approximately 2 x 104 cells/well. DNA samples (10 ng) were used in PCRs with BAT26, BAT25, or SMT15 primers (2 pmol/µl), dNTPs (200 mM) in a reaction buffer containing 0.5 U of Taq polymerase (Perkin-Elmer). Following the initial denaturation (95°C for 2 min), BAT26 and BAT25 were amplified by 35 cycles of 60 s at 95°C, 60s at 55°C, and 60 s at 72°C, followed by 10 min at 72°C. SMT15 was amplified by using a four-stage protocol (49) as follows: four cycles of 30 s at 95°C, 30 s at 64°C, and 2 min at 70°C; four cycles of 30 s at 95°C, 30 s at 61°C, and 2 min at 70°C; four cycles of 30 s at 95°C, 30 s at 58°C, and 2 min at 70°C; and four cycles of 30 s at 95°C, 30 s at 55°C, and 2 min at 70°C. Amplification products (10 µl) were digested with 0.4 U of T4 DNA polymerase (Roche) for 30 min at 37°C, denatured in deionized formamide for 2 min at 95°C, and analyzed with an ABI Prism 310 automatic sequencer.
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RESULTS |
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hMTH1 expression reduced the mutator effect in msh2-/- MEFs. There was an inverse relationship between hMTH1 expression and the hprt mutation rate (Table 1). The rate in msh2-/- MEFs was 3.1 x 10-6 mutations/cell/generation (mean of the three independent determinations), a value 25-fold higher than rates in wild-type mouse cells (16). This value is compatible to the reported 10- to 20-fold increase in spontaneous mutation rates in msh2-/- mouse tissues (4). Modest hMTH1 overexpression (around 10 times the endogenous level) in the pooled transfectants and in clone 2 halved this to 1.4 x 10-6 mutations/cell/generation. The 50-fold-enhanced hMTH1 expression in clone 5 was associated with a 17-fold reduction to 0.18 x 10-6 mutations/cell/generation. This is close to the calculated values for wild-type mouse cells (16). (We were unable to calculate mutation rates in msh2+/+ MEFs because of multiple copies of the hprt gene.) Transfection with the empty vector did not alter the hprt mutation frequency, and Western blot analysis indicated that all hMTH1-expressing clones remained msh2 defective (data not shown).
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hMTH1 and the hprt mutational spectrum.
hMTH1 hydrolyzes several oxidized purine dNTPs, including 8-oxodGTP, 2-oxodATP, and 8-oxodATP (20). In order to investigate the contribution of these oxidized precursors to spontaneous mutagenesis in MMR-defective cells, we compared spontaneous hprt mutational spectra in msh2-/- MEFs and hMTH1-overexpressing clone 5. Independent mutants from both cell lines were sequenced. Table 2 shows the frequency and rate of each type of mutation for each cell line. As expected for MMR-deficient cells, frameshifts were the major single mutational type in msh2-/- MEFs. They comprised more than one-third of all mutations (12 of 33; 36.4%). Base substitutions comprised around 60% of the total and were equally distributed between transitions and transversions (27.2 and 33.4%, respectively). There was a significant predominance of AT GC over other transitions (8 of 33 mutations; 24.2%), whereas the four possible transversions were approximately equally represented.
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G · C transitions 44.1-fold. There was a more modest effect on the rate of transversions, which was reduced only 8.9-fold. AT TA and GC TA mutations were particularly affected, whereas there was a surprisingly minimal impact on A C transversion rates, which were reduced only 3.2-fold.
The type and location of the mutation, the surrounding sequence, and the resulting amino acid change are summarized in Table 3 and Fig. 2. Base substitutions appeared to be randomly distributed, although there was some clustering, e.g., the four base substitutions at position 581 in clone 5. In a single case (mutant 53), a double mutation, formed by two TA GC transversions within three base pairs, was found in clone 5. In contrast, essentially all frameshifts were located within the run of six consecutive guanines at positions 207 to 213, which are a known frameshift hot spot in MMR-defective cells (30). Within this hot spot, all of the mutations scored were -1 deletions. A single +G frameshift was observed in clone 5, and it was located in a nonrepetitive sequence. We conclude that overexpression of hMTH1 reduced the rate of -1 frameshift mutations within the acknowledged G6 target by 34-fold. Thus, improved dNTP pool sanitation has a profound effect on the incidence of the type of mutations that are regarded as signatures of MMR deficiency. The striking reduction in frameshifts in the G6 target suggests that incorporation of 8-oxodGMP influences the generation of these mutations in an MMR-deficient cell.
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We also examined the effect of hMTH1 on MSI in the human prostatic cancer cell line DU145. This MMR-defective cell line has a profound mutator phenotype and bears mutations in both the hMLH1 and hMSH3 MMR genes (9). The BAT26 mutation rate was reduced 1.3-fold by 10-fold-increased hMTH1 activity in a clonal isolate (DU145/clone 1). More strikingly, hMTH1 expression dramatically altered the nature of the changes at BAT26. All (38 of 38) BAT26 mutations in DU145 were single A deletions. In contrast, there were only 2 deletions (of 27) among DU145/clone 1 mutations. This indicates that hMTH1 has a selective effect in reducing -1-base changes. This possibility was explored further by analyzing the SMT15 locus, which contains a run of eight G's. Increased hMTH1 expression in DU145/clone 1 reduced the mutation rate at this locus 10-fold (Fig. 3C). Again, all of the changes in DU145 were -G deletions. Examples of changes at BAT26 and SMT15 are shown in Fig. 3D. Thus, BAT26 and SMT15 provide clear evidence that oxidized NTPs have a significant influence on MSI. This can affect both A and G repeat microsatellites. The stability of BAT25 indicates that exceptions do exist and that additional factors, such as surrounding sequences, may also play a part. The selective effect of hMTH1 expression in reducing the frequency of -1-base deletions is particularly noteworthy.
Thus, hMTH1 expression affects two of the defining characteristic of MMR-defective cells. It reduces HPRT mutation rates. It also significantly ameliorates MSI at some, but not all, loci by preventing -1-base deletions. We conclude that the pool of oxidized purine dNTPs significantly influences the mutator phenotype of MMR-defective cells.
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DISCUSSION |
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The mutational spectrum in msh2-/- MEFs that express a high level of hMTH1 provides important clues to the events underlying their mutator phenotype. The range of hMTH1 substrates includes 8-oxodGTP, 2-oxodATP, and 8-oxodATP (20). One important implication of our data is that incorporation of both oxidized guanine and adenine contributes significantly to the mutator effect of MMR-defective cells. By comparison to 8-oxoadenine (47), 8-oxoG and 2-oxoA are both considered to be highly mutagenic. The pairing properties of 2-oxoA are particularly promiscuous, and it can direct incorporation of dCMP or dAMP to produce AT GC transitions and AT TA transversions, respectively (28). The most dramatic effect of hMTH1 expression was on the last two classes of base substitutions. AT GC and AT TA mutation rates decreased 44- and 62-fold, respectively. The most straightforward explanation for these findings is that during replication, 2-oxodAMP is incorporated opposite T. In the absence of MMR, the oxidized purine persists and forms the premutagenic mismatches 2-oxoA · C and 2-oxoA · A in the following round of replication (Fig. 4). It is interesting that a large increase in mutations at A · T base pairs is the major factor in the increased mutator phenotype of thymic lymphomas that arise in msh2-/- mice (50).
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TA transversions (24, 38). Consistent with a simple reduction in the level of 2-oxodAMP incorporation, hMTH1 expression also prevented this type of mutation. We note, however, an alternative route by which these mutations may arise that is also susceptible to modulation by hMTH1. GC TA transversions are signature mutations of template 8-oxoG. They could arise at persistent 8-oxodGMP that was incorporated in a previous replication round (Fig. 4). The contribution of persistent 8-oxodGMP is likely to be minimized by BER mediated by Ogg-1 and Myh, both of which are fully operative in these MMR-defective cells. We suggest that reversing the potentially mutagenic incorporation of 2-oxodAMP is a previously unrecognized function of MMR.
The minimal impact of hMTH1 expression (only threefold) on AT CG transversions is surprising. These mutations arise via the A · 8-oxoG mismatches that are an acknowledged product of 8-oxodGMP incorporation in in vitro replication systems (40). In addition, the mutator phenotype of E. coli mutT mutant strains is largely due to increases in this class of transversions (2). The modest effect of hMTH1 overexpression suggests that the majority of AT CG transversions in an MMR-defective background are the consequence of A · G mismatches.
One of the most significant findings is the dramatic reduction in -G frameshifts in the G6 tract of the hprt gene. This identifies oxidized dNTPs as a major contributor to frameshift mutagenesis in these msh2-/- cells—a finding that is consistent with the reported phenotype of mth1 knockout mice. Although these animals do not have an overt mutator phenotype, they do display an increased frequency of -A frameshifts in mononucleotide runs of the transgenic rpsL gene (19). We conclude that, in addition to causing base substitutions, oxidized dNTPs make a significant contribution to the production of -1-base frameshifts that are normally corrected by the mouse MMR system.
Increased hMTH1 expression in the two MMR-deficient human cell lines provided further evidence that oxidized dNTPs contribute to the mutator effect of repair-defective cells—and that they particularly influence frameshifts. Although we were unable to isolate transfectants with high hMTH1 expression, a modest level had a significant impact on the phenotype of both DLD-1 and DU145. The HPRT mutation rate in DLD-1 was reduced between two and threefold. Since the majority of HPRT- mutations in these hMSH6-deficient cells are base substitutions—with a relatively minor contribution from frameshifts (5, 34)—these data are consistent with hMTH1 preventing base substitutions in human HPRT as it does in its mouse counterpart. hMTH1 expression in DLD-1 also provided evidence of a significant impact on frameshifts—and by implication on MSI. The mutation rate at BAT26 was reduced about threefold. Although hMTH1 expression in hMHL1-defective DU145 did not produce a measurable decrease in the overall HPRT mutation rate (unpublished observation), it confirmed the influence on frameshifts. Although the decrease in the mutation rate at BAT26 was modest, it was accompanied by a 10-fold reduction in the rate in the G8 tract of SMT15. There were also important qualitative changes. Loss of the hMLH1 (but also of hPMS2 or hMSH2) MMR proteins in human tumor cell lines leads to an accumulation of +G frameshifts in the G6 target sequence of the HPRT gene (5, 39, 22). hMTH1 expression in the human cell lines, as in the msh2-/- MEFs, selectively affected minus frameshifts. This was apparent at both BAT26 and SMT15. Thus, oxidized dNTPs contribute to the generation of single-base deletions in repetitive mononucleotide sequences of MMR-defective human and mouse cells.
These findings have two important implications. The first concerns the designation of MSI. There are two An mononucleotide runs in the recommended panel of microsatellites (8). Our findings indicate that instability at BAT26 is likely to be influenced by changes in oxidative metabolism that increase steady-state levels of oxidized purine dNTPs. Secondly, the selective influence of oxidized dNTPs on -1 deletions has implications for the measurement of mutation rates. HPRT provides a particularly good example. In MMR-defective mouse cells, most hprt mutations are -G frameshifts in a G6 sequence. The same G6 sequence is a target in MMR-defective human cells, but in this case, mutations are overwhelmingly +G. Our data indicate that changes in oxidized purine dNTP levels have a more profound influence on -1-base alterations than on +1-base changes. The effect on mutation rates in MMR-defective cells of deranged oxidative metabolism and changes in steady-state oxidized purine dNTPs will therefore depend on the particular genetic target. We note that our data indicate the involvement of oxidized dNTPs in MSI at mononucleotides. Their influence on dinucleotide repeats remains undefined.
The oxidized dNTP that influences frameshifts in BAT26 remains unidentified. The different behaviors of BAT26 and BAT25 are, however, consistent with some of the known properties of 2-oxodATP. 2-OxoA in a double-stranded vector replicated in COS-7 cells induces -A deletions. Importantly, this effect was extremely dependent on both the sequence context of the modified purine and the orientation of its replication. More frameshifts were introduced by lagging-strand 2-oxoA (28). Effects on lagging-strand replication offer a plausible explanation for our finding that oxidized purine dNTPs favor the production of -1-base deletions and may contribute to the qualitative difference between mouse (predominantly -1 changes) and human (predominantly +1 changes) HPRT frameshifts (Fig. 5). In human cells, an origin of replication is located in the first intron of HPRT (13) and the G6 sequence is replicated by the lagging-strand DNA polymerase. In the mouse, the origin and direction of replication of the G6 tract in the third exon are not known. We propose that replication of the G6 target sequence in mouse cells is in the direction opposite to that of its human counterpart. Minus frameshifts might then arise in the msh2-/- mouse cells as a consequence of slippage of the C-containing template strand caused by 8-oxoG in the lagging daughter DNA strand during replication of the C6 tract. This is consistent with the increased propensity of the lagging-strand DNA polymerase to produce frameshifts compared to the more processive leading-strand polymerases (21, 33). It is also compatible with the acknowledged tendency of mammalian DNA polymerases to produce -1 frameshifts more frequently in runs of template pyrimidines owing to the relatively poor stacking of pyrimidines compared to purines (32). In human cells, the G6 sequence is replicated by the lagging-strand DNA polymerase and +1 deletions predominate. It is possible that the direction of replication might influence the type of frameshift intermediates generated by oxidized dNTPs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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M.T.R. and M.F.B. contributed equally to this work.
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and are single bases unless otherwise indicated. +g is a single-base insertion. The asterisk indicates a double mutant.
