Carcinogenesis, Vol. 20, No. 7, 1293-1301,
July 1999
© 1999 Oxford University Press
Carcinogenesis |
Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line
1 Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA,
2 CFSAN, Food and Drug Administration, Washington, DC, USA and
3 Laboratory of Molecular Genetics, National Institute of Aging, Baltimore, MD, USA
We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12–140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis.
4 Present address: Division of Biological Sciences, Institute of Environmental and Biological Sciences, Lancaster University, Lancaster LA1 4YQ, UK
5 To whom correspondence should be addressed Email: kraemerk{at}nih.gov
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