A role for DNA polymerase V in G→T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5′-T [formula omitted]T sequence in E. coli

Abstract

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g. GC→TA, GC→AT, etc.) principally via its major adduct [+ta]-B[a]P- N 2-dG. Recent findings suggest that different lesion bypass DNA polymerases may be involved in different mutagenic pathways, which is the subject of this report. [+ta]-B[a]P- N 2-dG built into a plasmid in a 5′-T G ̄ T sequence gives approximately equal numbers of G→T and G→A mutations when host E. coli are UV irradiated prior to transformation, so this sequence context was chosen to investigate what DNA polymerases are involved in G→T versus G→A mutations. G→T mutations decline (>10-fold) if E. coli either are not UV-irradiated or are deficient in DNA polymerase V (Δ umuD/ C), demonstrating a role for damage-inducible DNA Pol V in a G→T pathway. G→T mutations are not affected by transformation into E. coli deficient in either DNA polymerases II or IV. While the work herein was in progress, Lenne-Samuel et al. [Mol. Microbiol. 38 (2000) 299] built the same adduct into a plasmid in a 5′-G G ̄ A sequence, and showed that the frequency of G→T mutations was similar in UV-irradiated and unirradiated host E. coli cells, suggesting no involvement by damage-inducible, lesion bypass DNA polymerases (i.e., not II, IV or V); furthermore, a role for DNA Pol V was explicitly ruled out. The easiest way to reconcile the findings of Lenne-Samuel et al. with the findings herein is if two G→T mutagenic pathways exist for [+ta]-B[a]P- N 2-dG, where sequence context dictates which pathway is followed. In contrast to the G→T mutations, herein G→A mutations from [+ta]-B[a]P- N 2-dG in the 5′-T G ̄ T sequence context are shown not to be affected by UV-irradiation of host E. coli, and are not dependent on DNA Pol V, or Pol II, Pol IV, or the damage-inducible, but SOS-independent UVM system. Published studies, however, have shown that G→A mutations are usually enhanced by UV-irradiation of host E. coli prior to the introduction of plasmids either site-specifically modified with [+ta]-B[a]P- N 2-dG or randomly adducted with (+)-anti-B[a]PDE; both findings imply the involvement of a lesion-bypass DNA polymerase. These disparate results suggest the existence of two G→A mutagenic pathways for [+ta]-B[a]P- N 2-dG as well, although confirmation of this awaits further study. In conclusion, a comparison between the evidence presented herein and published findings suggests the existence of two distinct mutagenic pathways for both G→T and G→A mutations from [+ta]-B[a]P- N 2-dG, where in each case one pathway is not damage-inducible and not dependent on a lesion-bypass DNA polymerase, while the second pathway is damage-inducible and dependent on a lesion-bypass DNA polymerase. Furthermore, DNA sequence context appears to dictate which pathway (as defined by the involvement of different DNA polymerases) is followed in each case.