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[Cancer Research 61, 3775-3780, May 1, 2001]
© 2001 American Association for Cancer Research

Regular Articles

Hypermutability to Ionizing Radiation in Mismatch Repair-deficient, Pms2 Knockout Mice1

Xiaoxin S. Xu, Latha Narayanan, Brady Dunklee, R. Michael Liskay and Peter M. Glazer2

Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8040 [X. S. X., L. N., B. D., P. M. G.], and Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon 97201-3098 [R. M. L.].


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA mismatch repair (MMR) has been shown to play a role in the cytotoxicity of ionizing radiation (IR), as cell lines established from MMR-deficient mice exhibit higher clonogenic survival after IR than do cell lines from wild-type littermates. To test whether this tolerance phenotype would render MMR-deficient animals hypermutable to IR, we compared IR mutagenesis of Pms2-deficient versus wild-type transgenic mice carrying a {lambda} shuttle vector for mutation detection. In Pms2 nullizygous animals, the mutation frequency in the supFG1 reporter gene was increased from 210 x 10-5 in untreated animals to 734 x 10-5 after 6 Gy of IR (an increase of 524 mutants per 105), whereas the frequency in wild-type mice increased from 1.9 x 10-5 to 10.2 x 10-5 (an increase of only 8.3 mutants per 105). Similarly, when the {lambda} cII gene was used as a reporter, the mutation frequency in nullizygous mice was increased from 16.3 x 10-5 to 42.3 x 10-5 after IR (an increase of 26.0 x 10-5), whereas the frequency in wild-type mice increased from 2.4 x 10-5 to 9.4 x 10-5 (an increase of only 7.0 x 10-5). The pattern of IR-induced mutations in the MMR-deficient animals was notable for single bp deletions and insertions in mononucleotide repeat sequences, along with a slight increase in transversions. Overall, these results suggest that MMR-deficiency confers hypermutability to IR, and that much of this hypermutability can be attributed to induced instability of simple sequence repeats. Hence, MMR influences not only the survival but also the mutability of cells in response to IR.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammalian cells, there are several homologues of the Escherichia coli mutS and mutL DNA MMR3 genes, including Msh2, Msh3 and Msh6 and Mlh1, Pms1, and Pms2, respectively (reviewed in Refs. 1 and 2 ). Germ-line mutations in a number of these genes have been linked to hereditary cancers of the colon and other sites (1, 2, 3) . Biochemical and genetic studies have revealed that heterodimers formed between MSH2 and MSH6 (MutS{alpha}), or MSH2 and MSH3 (MutSß) are required for the recognition of single bp mismatches, small insertions/deletions, and single-stranded DNA loops, with some overlapping and some divergent specificities. The subsequent removal of these mismatched bases relies on a heterodimer formed by MLH1 and PMS2. Transgenic knockout mice have been generated to study the relationship between individual MMR proteins and cancer development. It has been shown that Mlh1 and Msh2 nullizygous mice develop intestinal adenomas, lymphomas, adenocarcinomas, and, to a lesser extent, skin tumors and sarcomas (4 , 5) . However, most of the cancers in Pms2 nullizygous mice are lymphomas (6) . More recently, the cancer predispositions of Msh3 and Msh6-deficient mice have been evaluated (7 , 8) .

Because the MMR factors have a fundamental role in the correction of replication errors, genetic instability was initially presumed to arise in MMR-deficient cells simply from the failure to fix such errors. However, a number of studies have implicated MMR factors in the response of mammalian cells to DNA damage. For example, certain MMR proteins, particularly MSH2 and MLH1, appear to have roles in transcription-coupled repair (9) , recombination (10) , and cell cycle regulation (11) . On the basis of work in bacteria suggesting that MMR can influence cellular survival after alkylation damage (12) , studies in mammalian cells have shown that MMR deficiency confers tolerance to alkylating agents (13 , 14) . Other studies have shown that loss of MMR is an important mechanism of resistance to a variety of clinically important drugs such as cisplatin (15 , 16) . To explain these observations, it has been proposed that MMR proteins can recognize and bind to various types of lesions in DNA (17 , 18) and initiate so-called futile cycles of repair that may be deleterious to the cell (19) . In addition, MMR-mediated recognition of DNA damage may also participate in a signal transduction pathway leading to apoptosis (20 , 21) .

Previously we had demonstrated a role for several of the MMR factors, including MSH2, MLH1, and PMS2, in the cytotoxicity of IR (22) . Our results indicated a small but statistically significant increase in clonogenic survival of MMR-mutant cells compared with wild type after IR exposure. Our initial work was carried out using immortalized fibroblasts derived from Msh2, Mlh1, or Pms2 nullizygous mice and from wild-type littermates. Subsequently, DeWeese et al. (23) , using embryonic stem cells from Msh2 knockout mice, reported that, at low radiation dose rates, the survival differences between wild-type and Msh2-deficient cells were even larger than the differences seen at high dose rates. In addition, Zhang et al. (24) found that IR-induced apoptosis was reduced in Msh2-nullizygous mouse embryonic fibroblasts compared with wild-type. In more recent work, we extended our initial observations in immortalized cells to primary fibroblasts from Pms2-deficient mice, finding that such cells show less IR-induced apoptosis than do wild-type ones (25) .

Our observation of increased IR survival in MMR-deficient cells led us to propose that IR-induced oxidative damage, like alkylation damage, was subject to MMR recognition and that the damage tolerance phenotype in MMR-deficient cells therefore also extended to oxidized bases (22) . On the basis of this model, we hypothesized that MMR-deficient cells would show hypermutability to IR. Here, we have tested this hypothesis by examining IR mutability in either wild-type or Pms2-deficient transgenic mice carrying two recoverable mutation reporter genes (supFG1 and cII). We find that Pms2-deficient animals are hypermutable to IR compared with wild-type at both reporter loci, with a pattern of induced mutations notable for single bp deletions (plus a few insertions) within repeated sequences. These results suggest that genetic instability in MMR-deficient cells can arise not only from uncorrected replication errors but also from an altered response to IR-induced DNA damage. In addition, the results provide additional support for the emerging concept that certain classes of DNA damage can destabilize repeated sequences (26) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic Mice.
Generation of wild-type and Pms2-deficient mice carrying a recoverable {lambda}supFG1 mutation reporter vector has been described (27) . Briefly, 3340 mice carrying 15 copies of the {lambda}supFG1 shuttle vector DNA at a single chromosomal locus were crossed with mice heterozygous for targeted disruption of the Pms2 gene. Mice carrying both the {lambda}supFG1 vector and the Pms2 null allele were interbred to yield littermates either wild-type, heterozygous, or nullizygous for Pms2, while also carrying the {lambda}supFG1 vector DNA. Although the Pms2 knock-out mice were originally derived using D3 embryonic stem cells of 129/Sv mouse origin (6) , the mice have been back-crossed into the C57BL/6 mouse background. The Pms2 genotype of the mice was determined by the presence or absence of the targeted insertion by PCR amplification of a region in the Pms2 gene, as described (6) . The presence of the supFG1 gene in the mice was also confirmed by PCR amplification, as described (27) .

Irradiation.
Wild-type and Pms2-deficient mice 12 weeks of age were exposed to total body irradiation with a dose of 600 cGy using a 137 Cs irradiator at a dose rate of 225 rad/min. Mice were sacrificed 7 days after irradiation, and genomic DNA was prepared from mouse skin as described previously (27) .

Mutagenesis Assay.
Mutagenesis in the transgenic mice was assayed using the {lambda} shuttle vector system. High molecular weight genomic DNA was isolated from mouse skin as described previously (27) . {lambda} vector rescue from the mouse DNA was carried out using {lambda} in vitro packaging extracts (28 , 29) . Packaging extracts were made as described previously (28) , using the E. coli lysogen, NM759 [E. coli K12 recA56 {Delta}(mcrA) e14° {Delta}(mrr-hsd-mcr) ({lambda}imm434 cIts b2 red3 Dam15 Sam7)/{lambda}] instead of BHB2690 for the preparation of the sonicate extract (29) . This lysogen produces extracts that are deficient in methyl-directed restriction activity that would otherwise degrade DNA methylated in the mammalian pattern and reduce the yield of rescued phage (29 , 30) . The mouse DNA was incubated in the {lambda} in vitro packaging extracts at a concentration of 0.05 µg/µl for 3 h at 30°C. The packaged phage were diluted in 10 mM Tris (pH 8.0) and 5 mM MgCl2, adsorbed to PG901 [E. coli C1a lacZ125 (am)], and plated in 0.4% top agar on Luria-Bertani plates in the presence of X-Gal (1.6 mg/ml) and isopropyl-1-thio-ß-D-galactopyranoside (1.3 mg/ml), as described (28) . Phage with functional supFG1 genes suppress the nonsense mutation in the host bacteria ß-galactosidase gene, allowing synthesis of an active enzyme capable of metabolizing X-Gal, thereby yielding blue plaques. Phage with inactivating supFG1 mutations produce colorless plaques. For cII gene mutation detection, the in vitro packaged {lambda} phage were adsorbed to an E. coli G1250 hflA ::Tn5 hflB29 (31) , and grown at 24°C for 48 h. At this temperature in this host, wild-type phage form lysogens and so do not produce detectable plaques, whereas phage with inactivating mutations in the cII gene will not form lysogens and are identified by lytic plaque production. The total number of packaged phage is determined by plating on E. coli G1250 at 37°C, which allows lytic plaque production regardless of cII status because of the cI857 temperature-sensitive {lambda} repressor allele carried in the phage vector.

Sequence Analysis.
After plaque purification, the supFG1 genes in the mutant phage were amplified by PCR and sequenced as described previously (27) . The cII genes in the mutant plaques were amplified as described previously (31) , with some modification. The primers used for PCR were 5'-CGC TCT TAC ACA TTC CAG CC-3' (C2P1XU) and 5'-CTG CCA CAT TAC GCT CCT GTC C-3' (C2P3XU). The PCR reaction was denatured at 95°C for 2 min and then run for 40 cycles at 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min. The PCR products were purified using a PCR purification kit (Qiagen) according to the manufacturer’s directions, and sequenced using primer C2P1XU via automated methods as described (27) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IR-induced Mutation Frequencies in Wild-Type and Pms2 Null Mice.
In previous work, we examined clonogenic survival of immortalized mouse fibroblast cell lines derived from MMR nullizygous and wild-type animals subsequent to exposure to IR (22) . The MMR-deficient cell lines, lacking either Msh2, Mlh1, or Pms2, all showed increased survival after IR compared with wild-type cell lines. In more recent work, we found that Pms2 nullizygous primary cells show a decreased susceptibility to IR-induced apoptosis compared with primary cells from wild-type littermates (25) .4 One prediction from this phenotype of IR tolerance is that mouse cells or mice deficient in MMR will show increased mutagenesis after IR.

To test this hypothesis, we established a series of transgenic mice carrying the {lambda}supFG1 mutation reporter vector that were also either wild-type, heterozygous, or nullizygous for Pms2 (27) . Wild-type and Pms2-deficient mice 12 weeks of age carrying the {lambda} shuttle vector were irradiated with 600 cGy of IR as a total body dose, and the mutation frequencies were determined in both the supFG1 and cII reporter genes. After irradiation, a period of 7 days was allowed for mutation expression, based on the work of Winegar et al. (32) who reported a time-course analysis of IR-induced mutagenesis in wild-type C57Bl/6 mice carrying a {lambda}lacI reporter vector.

The analysis focused on phage vectors rescued from the skin of both the wild-type and Pms2-deficient mice. For each genotype, mutagenesis was assayed in both irradiated animals and unirradiated controls. In wild-type mice, the mutation frequency in the supFG1 reporter gene was increased by IR from 1.9 x 10-5 to 10.2 x 10-5 (an increase of 8.3 per 105), whereas in Pms2 nullizygous animals, the supFG1 mutation frequency increased from 210 x 10--5 to 734 x 10-5 after IR (a difference of 524 per 105; Table 1Citation and Fig. 1Citation ). Similarly, in the {lambda} cII reporter gene, the mutation frequency in wild-type animals increased from 2.4 x 10-5 to 9.4 x 10-5 (a difference of 7 x 10-5) after IR; whereas in Pms2-deficient mice, the mutation frequency in cII increased from 17.2 x 10-5 to 42.3 x 10-5 (an increase of 25.1 x 10-5; Table 2Citation and Fig. 2Citation ). Note that the differences in absolute frequency, rather than fold-increases, are given. These absolute differences reflect directly the extent of additional mutagenesis induced by IR treatment in each genetic background.


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  		Table 1 Spontaneous and IR-induced mutation frequencies in the supFG1 gene in wild-type, heterozygous, and Pms2 nullizygous mice

 


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  		Fig. 1. Comparison of IR-induced mutagenesis in the supFG1 reporter gene in wild-type and Pms2-deficient mice. The animals were irradiated with 600 cGy, and the frequency of mutations was determined in mouse skin 7 days after irradiation. The last two columns indicate the magnitude of the difference between the unirradiated and irradiated samples for both the wild-type and the Pms2 nullizygous mice. Error bars, SE of proportions.

 

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  		Table 2 Spontaneous and IR-induced mutation frequencies in the cII gene in wild-type, heterozygous, and Pms2 nullizygous mice

 


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  		Fig. 2. Comparison of IR-induced mutagenesis in the cII reporter gene in wild-type and Pms2-deficient mice. The animals were irradiated with 600 cGy, and the frequency of mutations was determined in mouse skin 7 days after irradiation. The last two columns indicate the magnitude of the difference between the unirradiated and irradiated samples for both the wild-type and the Pms2 nullizygous mice. Error bars, SE of proportions.

 
As in a previous study comparing genetic instability in a series of reporter genes (33) , the supFG1 gene was found to be particularly prone to mutagenesis in the absence of MMR, as expected from the two long mononucleotide repeat tracts in the coding sequence (27) . However, although the absolute values for supFG1 and cII are different, reflecting the relative sensitivities of the reporter genes, both the supFG1 and cII results indicate that IR induces substantially more mutations/rad in the Pms2 nullizygous animals than in the wild-type animals.

In addition, note that the heterozygous animals had frequencies of spontaneous and induced mutations similar to those of the wild-type ones.

Mutation Spectra.
The pattern of cII gene mutations induced by IR in the Pms2-null mice was determined and compared with that found in Pms2-null animals in the absence of irradiation (Fig. 3Citation and Table 3Citation ). In untreated Pms2-deficient mice, a large percentage of the mutations were single bp deletions (and some insertions) within mononucleotide repeat sequences, consistent with previous studies (27 , 34) . The base substitution mutations in this case were all transitions; no transversions were seen. In IR-treated animals, there was a marked preponderance of single bp deletions (49%) within mononucleotide repeat sequences. In particular, a hot spot for single bp deletions was observed within a 6-A:T-bp run, and a number of single bp deletions (along with a few insertions) were also found in a run of 6 G:C bp. In addition, there was an increase in the frequency of transversions compared with the unirradiated sample; overall, transversions accounted for only 18% of the mutations. These cII spectra were compared using a statistical package (35) , and the differences were found to be statistically significant (P < 0.0015). In the supFG1 gene, a similar pattern was seen; 73% of the IR-induced mutations in the Pms2 animals were either single bp insertions or deletions in one of two G:C bp stretches of length 7 and 8 (data not shown). The higher absolute mutation frequencies in the supFG1 reporter likely reflect the hypermutability of these sites.



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  		Fig. 3. Sequences of mutations found in the cII reporter gene rescued from Pms2-deficient mice. The wild-type sequence of cII gene is presented, with the mutations induced by IR listed above the cII sequence and the spontaneous mutations listed below. Single bp insertions and deletions are indicated by + and - signs, respectively. One mutation consisted of a 3-bp deletion and is indicated by parentheses.

 

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  		Table 3 Spectrum of cII mutations in Pms2-deficient mice

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this work, we have examined IR-induced mutagenesis in wild-type and MMR-deficient animals lacking the mutL homologue gene, Pms2. We find that the Pms2 nullizygous mice are hypermutable to IR, as measured in two different mutation reporter genes, supFG1 and cII. In the Pms2-deficient mice, the frequency differences between IR-induced mutagenesis and spontaneous mutagenesis were 524 per 105 in supFG1 and 26 per 105 in cII. In contrast, in wild-type animals the differences were only 8 per 105 and 7 per 105, respectively. Hence, the Pms2 nullizygous animals are more susceptible to mutation after IR damage.

Hypermutability to IR was predicted on the basis of work indicating that MMR-deficient cells show increased survival and decreased apoptosis after IR (22, 23, 24, 25) . We reasoned that the tolerance phenotype for IR-damage would mean that an increased number of damaged cells would survive irradiation and that they would do so with a potentially higher level of DNA damage. This would lead to an increase in induced mutagenesis.

This same reasoning was applied to the phenomenon of alkylation tolerance in MMR-deficient cells, and a hypothesis of hypermutability to alkylation damage was tested by Andrew et al. (33) . They found that Msh2-nullizygous mice carrying a {lambda}lacI vector were hypermutable to the alkylating agent, MNU. Their data and ours are summarized and presented together in Fig. 4Citation . Mutagenesis in the wild-type and MMR-deficient animals is plotted as a function of MNU and IR dose. Note that the lines for the Msh2 null and Pms2 null animals have greater slopes than those for the wild-type animals, indicative of the phenomenon of hypermutability. The similarity found in these data sets suggests that similar mechanisms may underlie the MNU and IR damage tolerance and hypermutability.



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  		Fig. 4. Comparison of MNU and IR-induced mutagenesis in wild-type and MMR-deficient mice. A, MNU-induced mutagenesis in the lacI reporter gene in small intestine of Msh2-deficient mice. B, MNU-induced mutagenesis in the lacI reporter gene in thymus of Msh2-deficient mice. C, IR-induced mutagenesis in the cII reporter gene in skin of Pms2-deficient mice. D, IR-induced mutagenesis in the supFG1 reporter gene in skin of Pms2-deficient mice.

 
It has been suggested that the MMR complex can recognize certain types of base damage, including alkylation damage (17 , 18 , 36) and oxidative damage (37) . In one model, recognition of base damage may lead to futile cycles of repair, in which the MMR complex initiates repair but may incorrectly excise and resynthesize the undamaged strand, "recreating" the damaged substrate for another round of repair, in a process that may be lethal to the cell (19) . In another model, it has been suggested that the MMR repair complex recognizes certain classes of DNA damage and directly initiates a signal transduction pathway leading to apoptosis (21) . In either case, cells lacking MMR would suffer less cytotoxicity from the base damage, would survive with a higher level of damage, and would consequently be at risk to incur more mutations.

On the other hand, some studies have suggested that certain MMR factors may have a role in promoting transcription-coupled repair of DNA damage (9 , 38, 39, 40) . As a result, the lack of these MMR factors would reduce the repair capacity of the cells, at least for transcribed genes. Thus far, MSH2 (but not PMS2) has been implicated in promoting BER of oxidative damage (39) . Reduced BER in MMR-deficient cells would tend to cause increased sensitivity to base damage, and this phenomenon may be a countervailing factor that would serve to reduce the apparent damage tolerance phenotype. However, the reduced repair would still contribute to the hypermutability, but by a different mechanism.

The pattern of mutations induced by IR in the Pms2-nullizygous mice was notable for a preponderance of single bp deletions in short mononucleotide runs, a pattern that is also seen in unirradiated Pms2-deficient animals (27 , 34) . In contrast, in wild-type mice and cells exposed to IR, the spectra of induced point mutations typically includes a much higher proportion of base substitutions, with a particular increase in some classes of transversions (32 , 41 , 42) . If the increased mutagenesis in Pms2-dificient mice were attributable only to the increased survival of cells with IR damage, a mutation pattern similar to that seen in irradiated wild-type cells would be expected. However, except for a small increase in transversions, most of the increase in mutagenesis subsequent to IR in the Pms2-deficient mice can be accounted for by single bp deletions in mononucleotide repeat sequences. This atypical pattern suggests that the metabolism of IR-damaged DNA is altered in MMR-deficient cells. It may be that the IR-generated oxidative base damage destabilizes the duplex, leading to a greater degree of template dislocation and slippage during replication and possibly during some types of repair synthesis. In wild-type cells, such slippage events would be efficiently corrected by the MMR pathway; however, in MMR-deficient cells, the slippage errors persist unrepaired, leading to deletion and insertion mutations. This problem is compounded by the damage tolerance phenotype, in which more cells survive with unrepaired damage, and by the possibility of reduced BER in the absence of MMR, so that there is more persisting damage around to destabilize the DNA and promote slippage.

Support for this model comes from the work of Jackson et al. (26) , who demonstrated the instability of repeated sequences in E. coli in the face of oxidative damage. Interestingly, they found, in contrast, that alkylation damage caused by N-methyl-N'-nitro-N-nitrosoguanidine did not lead to an increase in repeat sequence instability (26) . Consistent with this latter observation, Andrew et al. (33) did not find a substantial increase in mutations within mononucleotide runs in the pattern of MNU-induced mutations in Msh2 nullizygous mice. Rather, they found that the hypermutability caused by MNU in the MMR-deficient animals could be accounted for primarily by G:C to A:T transitions at GpG sites (33) .

The preponderance of single bp deletions, rather than insertions, in the IR-induced mutation spectrum in the Pms2-null animals is also notable. In work examining spontaneous mutation patterns in Pms2, Mlh1, and Msh2 null mice, we had observed previously that the ratio of deletions to insertions is highest in Pms2 null mice (34) . Contraction mutations within mononucleotide repeat sequences are thought to arise from DNA slippage events causing single-strand loop formation on the template strand. Pms2 deficiency may specifically impair correction of such intermediates, leading to an increase in deletions.

In yeast, there is evidence that, in the absence of the OGG1 glycosylase, MMR plays an important role in suppressing mutagenesis attributable to oxidative DNA damage that arises endogenously from aerobic metabolism (37) . The yeast MSH2/MSH6 heterodimer was shown to bind to DNA containing 8-oxo-G, and it was suggested that the MMR pathway serves to correct errors that come about when DNA containing 8-oxo-G is replicated, leading to 8-oxo-G:A misincorporation events (37) . This role for MMR could theoretically contribute to hypermutability to IR in MMR-deficient mice, because 8-oxo-G is one of the major types of base damage caused by IR. However, we did not see a significant increase in the G:C to T:A mutations that would be expected to arise by this mechanism.

Another interesting aspect of the induced mutation data are that the heterozygous mice did not show hypermutability to IR. Because the human syndrome of hereditary nonpolyposis colorectal cancer typically involves heterozygous individuals who have inherited a single defective allele for one of several MMR genes (1 , 2) , the results presented here suggest that MMR heterozygotes are unlikely to be at increased risk of mutation, even when exposed to a high level of oxidative DNA damage. However, MMR-deficient cell lineages that may arise within such individuals (attributable either to a loss of heterozygosity on the chromosome level or to an acquired mutation in the wild-type allele) would be expected to exhibit hypermutability in response to oxidative damage, including the endogenous oxidative damage that results from aerobic metabolism. This would serve to promote further the accumulation of carcinogenic changes and would constitute another type of genetic instability that may contribute to colon cancer and other malignancies (43 , 44) .

In addition, the data presented here provide additional evidence that the MMR pathway plays an important role in the response of mammalian cells to IR. Although it is well established that base damage caused by IR is repaired by BER, and that IR-induced strand breaks are processed by a number of recombination and end-joining pathways, it is becoming clear that the MMR complex plays an important and multifaceted role in how cells process IR damage. This concept may have important implications for risk assessment and for understanding the response of normal and malignant cells to radiotherapy.


   ACKNOWLEDGMENTS
 
We thank J. A. Fritzell, J. Yuan, M. Zeng, L. Cabral, R. Franklin, and S. J. Baserga for their help.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the USPHS (NIH/National Institute of Environmental Health Sciences-5 R01 ES05775). X. S. Xu was supported by NIH Training Grant 5 T32 CA09259-17. Back

2 To whom requests for reprints should be addressed, Department of Therapeutic Radiology, Yale University School of Medicine, P. O. Box 208040, New Haven, CT 06520-8040. Phone: (203) 737-2788; Fax: (203)737-2630; E-mail: peter.glazer{at}yale.edu Back

3 The abbreviations used are: MMR, mismatch repair; IR, ionizing radiation; MNU, methylnitrosourea; BER, base excision repair. Back

4 Unpublished data. Back

Received 7/12/00. Accepted 2/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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