Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line

We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA seque nce and mismatch repair on mutagenesis in human cells. pLSC contains an int errupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRN A gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mu tations in pLSC was found to be similar after replication in either the hMS H6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6, However, the classes of plasmid muta tions showed distinct differences in the two cell lines. Single base deleti ons comprised 48% of the mutations in the 56 independent pLSC plasmids sequ enced 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 sequenc e, 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 muta tions in TK6 and MT1 cells. These were predominately single base substituti on mutations without a large increase in deletion mutations in the mononucl eotide run in pLSC, These data indicate that a mononucleotide poly(G) run p romotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutage nesis.