title: A role for DNA polymerase V in G --> T mutations from the major benzo[a]pyrene N2-dG adduct when studied in a 5'-TGT 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-N2-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-N2-dG built into a plasmid in a 5'-TGT 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 ((delta)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'-GGA 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-N2-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-N2-dG in the 5'-TGT 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-N2-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-N2-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-N2-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.
authors.affiliation: Biology Department, Boston University, Boston, MA 02215, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.forename: Kwang Young
authors.initials: KY
authors.lastname: Seo
authors.forename: Edward L
authors.initials: EL
authors.lastname: Loechler
mesh_terms: D001580:Benzopyrenes / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006147:Guanine; D016296:Mutagenesis / Q000235:genetics / Q000502:physiology*; D009153:Mutagens / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D013941:Thymine; D014466:Ultraviolet Rays
chemical_list: D001580:Benzopyrenes; D029968:Escherichia coli Proteins; D009153:Mutagens; D009841:Oligonucleotides; C478198:benzo(a)pyrene N2-dG adduct; D006147:Guanine; C120926:DNA polymerase V, E coli; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D013941:Thymine