Mutagenic effects of 5-formyluracil on a plasmid vector during replication in Escherichia coli

Purpose : 5-Formyluracil (5-foU) is a major derivative of thymine produced in DNA by ionizing radiation and various chemical oxidants. It has been previously shown that 5-foU in template DNA directs misincorporation of nucleotides by DNA polymerases during in vitro DNA synthesis. The present experiments were designed to understand the biological effects of 5-foU in vivo. Materials and methods : The modified base was incorporated site-specifically into the recognition site of restriction endonuclease Sal I (5'-GTCGAC) or Afl II (5'-CTTAAG) in vector plasmid pSVK3 and introduced the plasmid into Escherichia coli. Results : When the plasmids were replicated in E. coli, 5-foU caused mutations at the target sites. The induced mutation frequencies were 0.038-0.049%. Sequence analysis revealed that 5-foU preferentially caused T:A →C:G and T:A →A:T base substitutions and -1 deletions at the 5-foU site. 5-FoU also caused mutations at sites near the 5-foU. The alkA mutation did not affect the frequency of mutations in 5-foU-containing plasmids. Conclusions : The present experiments demonstrated that 5-formyluracil in DNA caused mutations in E. coli.