1 Department of Medical Genetics, University of Alberta, Edmonton,Alberta, Canada T6G 2H7 and
2 Centre for Molecular Medicine and Therapeutics and the Department of Medicine, University of British Columbia, Vancover, British Columbia, Canada V5Z 4H4
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Abstract |
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Abbreviations: DNA pol ; DNA polymerase
; MMR, mismatch repair; PCR, polymerase chain reaction.
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Introduction |
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A lack of DNA mismatch repair (MMR) has been shown to result in a mutator phenotype (2,3). MMR is a post-replicative repair system that acts on mispaired nucleotides as well as small insertion/deletion loops generated by misincorporation during DNA synthesis. This repair system is therefore instrumental in maintaining the fidelity of DNA. In the mouse, Msh2 is responsible for the initial recognition of a mispair and subsequent recruitment of additional proteins necessary for MMR. To study mammalian MMR, Reitmair et al. (4) generated homozygous Msh2/ mice by gene targeting technology. These mice were viable, but rapidly developed tumours, primarily lymphoid, starting at about 2 months of age. To study mutation frequency in the absence of MMR, Msh2/ mice were bred with transgenic mice carrying the lacI reporter gene (5,6). Using the lacI mutation detection assay, Msh2/ mice demonstrate a 10- to 15-fold increase in spontaneous mutation frequency as compared with Msh/+ or wild-type mice (6). DNA isolated from the Msh2/ thymic lymphomas revealed an even greater increase in mutational frequency, with values 3- to 17-fold higher than in non-tumour Msh2/ thymic tissue (7). Other Msh2/ tumours did not demonstrate such elevated mutation frequencies, but were similar to values seen in Msh2/ non-tumour tissues, suggesting that the increase in mutation frequency was specific to the thymic lymphomas. Interestingly, the lacI assay revealed closely clustered mutations that were not observed in non-tumour tissue or other Msh2/ tumours (7).
The greatly elevated mutation frequency in Msh2/ thymic lymphomas, the latency period before tumours appear and the closely clustered multiple mutations specific to the lymphomas all suggest subsequent mutations in additional `mutator' genes with an additive or synergistic mutator effect, have been acquired in MMR-deficient thymi. Aberrant replication is one such mechanism that has been shown to induce such a mutator phenotype (8). DNA replicative slippages as a result of a mutated DNA polymerase may account for the observed hypermutator effect and is therefore a likely hypothesis to explain the increase in mutation frequency. Thus, mutations in DNA polymerase genes that alter the enzyme fidelity could be responsible for the above characteristics of thymic lymphomas. For example, DNA polymerase ß mutations have been shown to result in a mutator phenotype associated with several forms of cancer (911). DNA polymerase (DNA pol
), is another potential polymerase that has an intrinsic 3'5' exonuclease activity and has been implicated in DNA repair (12,13). When DNA pol
is mutated, its catalytic properties are abnormal suggesting a potential mutator activity possibly resulting in a mutator phenotype (14). These results are consistent with the findings that Saccharomyces cerevisiae strains with mutations in the 3'5' exonuclease domain of DNA pol
are hypermutable (15). Supporting this result, Tran et al. have shown that an error-prone (or abnormal) DNA pol
results in a mutator phenotype in S.cerevisiae and that double Msh2//Pol
mutations have hypermutation frequencies above either mutation alone, suggesting that Msh2 and DNA pol d act synergistically (16). Da Costa et al. examined the 3'5' exonuclease domain of DNA pol
and found that three out of seven colon cancer cell lines contained a DNA alteration causing an amino acid change, suggesting that a mutated DNA pol
leads to tumorigenesis (17). Further implicating DNA pol
in tumorigenesis, Flohr et al. identified point mutations in the catalytic subunit of DNA pol
in human colon cancer cell lines and in primary human colon cancers (18). Additional support for the involvement of DNA pol
in Msh2/ tumorigenesis arises from the observation that, in the absence of MMR, some genes are non-random targets for mutations due to the presence of coding mononucleotide repeats of
5 bp. Several genes containing coding mononucleotide repeats have been identified as downstream target genes in MMR-deficient tumours. Coding mutations in such mononucleotide runs have been identified in BAX, caspase-5, IGFRII, transforming growth factor ß receptor II, MSH6 and MSH3 in microsatellite unstable (MSI+) colorectal and endometrial cancers (1924). DNA pol
contains six exons with mononucleotide runs of >5 bp and therefore justified a similar candidate gene screening approach assaying for a mutated DNA polymerase in murine MMR-deficient thymic lymphomas. Other DNA polymerases, such as DNA polymerase ß, display mutator phenotypes but are less likely candidates because they lack coding mononucleotide tracts. From the above data and in particular, the existence of six coding mononucleotide runs, we hypothesized that a mutated DNA pol
is a likely candidate mutator gene in Msh2/ murine thymic lymphomas. It is likely that a mutation in the catalytic subunit of DNA pol
would cause decreased DNA copying fidelity and so lead to increased genomic instability and progression to malignancy.
Mice lacking the DNA MMR gene, Msh2, have been developed as a model for studying the relationship between a mutator phenotype and development of cancer. Msh2/ mice develop thymic lymphomas with a hypermutator phenotype. We hypothesized that, in Msh2/ thymic lymphomas, an additional mutator gene (in conjunction with MMR loss) was responsible for this increased hypermutator effect.
To identify the possible role of the DNA pol catalytic subunit in the development of Msh2/ thymic lymphomas, all 26 exons of the gene were sequenced from genomic DNA in nine Msh2/ thymic lymphomas and two control (non-tumorous) liver and brain samples from wild-type mice. DNA was extracted from tissues as previously described (25). Intronic primers (26) (GenBank accession number AF024570) (Table I
) were used to amplify thymic lymphoma genomic DNA and control genomic DNA by polymerase chain reaction (PCR) (Table II
) and products were examined for mutations by 33P sequencing (Thermo Sequenase Cycle Sequencing, Amersham Pharmacia). Comparison of the two published DNA pol
catalytic subunit sequences revealed sequence differences at seven locations (Table III
). These differences in published sequence probably demonstrate sequencing errors of the cDNA or genomic DNA or differences in genetic backgrounds of the mice. The published genomic DNA sequence of DNA pol
(26) is from the 129SVJ mouse strain and the published cDNA sequence (12) is from the BalbC/9 strain. The Msh2 mice are on a mixed genetic background of BalbC and 129 mouse strains.
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DNA pol sequence from the thymic lymphomas revealed three locations where the sequence was different from both published sequences (exons 12, 13 and intron 17). One change was silent (exon 12), one was intronic (intron 17) and one resulted in an amino acid alteration (exon 13) (Table III
). All changes were confirmed by repeat sequencing. Within exon 3 at nucleotide position 1532, three of the nine tumours (tumours B, C and H) contain a T residue instead of a T residue as in the published sequence (Table III
) and at nucleotide position 4876 in exon 12, four of the nine tumours (tumours B, C, D and G) and both control tissues contain a T residue instead of a C residue as in the published sequences. The exon 12 alteration is silent. The intronic sequence alteration in intron 17 was seen in three tumours (tumours B, D and I) and in the controls; it is believed to be non-pathogenic as it does not alter the splice site junction of intron 17exon 17. Exon 13 is the only alteration resulting in a polymorphic amino acid change that differed from both the cDNA and genomic DNA published sequences. This alteration was seen in four of the nine tumours sequenced (tumours B, C, D and H). However, the alteration is not considered to be disease associated as two non-tumour wild-type control tissues demonstrated the same nucleotide sequence alteration (Table III
). Furthermore, this DNA alteration results in a conservative substitution of a serine replacing a glycine; both of these are neutral, uncharged amino acids, so this substitution is not predicted to result in altered protein function. Although this change is not likely to be associated with tumorigenesis, it is located adjacent to a conserved polymerization II (Pol II) domain of DNA pol
(12), so the close proximity of this polymorphism to the Pol II polymerization domain could subtly affect enzyme function or fidelity. Further functional analyses are required to confirm this possibility.
In the absence of MMR, mononucleotide repeats of 5 bp are prone to mutation through expansion and contraction of these repeats. Exons 3, 8, 16, 18 and 22 each contain mononucleotide repeats of
5 bp, which were hypothesized to be non-random locations for polymerase slippage. No mutations were seen at these locations, in normal or tumour DNA. Contrary to our hypothesis, no disease-associated mutations were found in the coding sequence of DNA pol
in the nine Msh2/ thymic lymphomas that were screened (Table III
), suggesting that polymerase slippages in the absence of MMR do not result in mutations of the mononucleotide tracts of DNA pol
.
Several polymorphic DNA alterations were seen in the tumours and controls (Table III). The identified changes seen in our lymphomas are unlikely to be disease associated as they were observed in both tumour and non-tumour control tissue and probably represent differences in the strain of mice used or errors in the published cDNA or genomic DNA sequences.
Evidence supporting the involvement of DNA pol in the development of Msh2/ thymic lymphoma and the presence of six coding mononucleotide repeats led us to sequence the 26 exons of the DNA pol
gene in nine Msh2/ thymic lymphomas and two wild-type controls from Msh2/ littermates. No lymphoma-associated mutations were found in the coding region of DNA pol
. Notably, the six coding mononucleotide repeat sequences were not mutated. The absence of repeat tract mutation indicates that the DNA pol
gene is not a non-random target for hypermutation-driven mutagenesis in Msh2/ murine thymic lymphomas. Continued investigation of other possible candidate genes, including other DNA polymerases such as DNA polymerase ß, is required to understand further the molecular events underlying the development of thymic lymphoma in the Msh2-deficient mouse.
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Notes |
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Acknowledgments |
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References |
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