8-OxodGuo and Fapy•dG Mutagenicity in Escherichia coli Increases Significantly when They Are Part of a Tandem Lesion with 5-Formyl-2'-deoxyuridine.

Tandem lesions, which are defined by two or more contiguously damaged nucleotides, are a hallmark of ionizing radiation. Recently, tandem lesions containing 5-formyl-2'-deoxyuridine (5-fdU) flanked by a 5'-8-OxodGuo or Fapy•dG were discovered, and they are more mutagenic in human cells than the isolated lesions. In the current study, we examined replication of these tandem lesions in Escherichia coli. Bypass efficiency of both tandem lesions was reduced by 30-40% compared to the isolated lesions. Mutation frequencies (MFs) of isolated 8-OxodGuo and Fapy•dG were low, and no mutants were isolated from replication of a 5-fdU construct. The types of mutations from 8-OxodGuo were targeted G → T transversion, whereas Fapy•dG predominantly gave G → T and G deletion. 5'-8-OxodGuo-5-fdU also gave exclusively G → T mutation, which was 3-fold and 11-fold greater, without and with SOS induction, respectively, compared to that of an isolated 8-OxodGuo. In mutY/mutM cells, the MF of 8-OxodGuo and 5'-8-OxodGuo-5-fdU increased 13-fold and 7-fold, respectively. The MF of 5'-8-OxodGuo-5-fdU increased 2-fold and 3-fold in Pol II- and Pol IV-deficient cells, respectively, suggesting that these polymerases carry out largely error-free bypass. The MF of 5'- Fapy•dG-5-fdU was similar without (13 ± 1%) and with (16 ± 2%) SOS induction. Unlike the complex mutation spectrum reported earlier in human cells for 5'- Fapy•dG-5-fdU, with G → T as the major type of errors, in E. coli, the mutations were predominantly from deletion of 5-fdU. We postulate that removal of adenine-incorporated opposite 8-OxodGuo by Fpg and MutY repair proteins is partially impaired in the tandem 5'-8-OxodGuo-5-fdU, resulting in an increase in the G → T mutations, whereas a slippage mechanism may be operating in the 5'- Fapy•dG-5-fdU mutagenesis. This study showed that not only are these tandem lesions more mutagenic than the isolated lesions but they may also exhibit different types of mutations in different organisms.