Mirror image stereoisomers of the major benzo[a]pyrene N2-dG adduct are bypassed by different lesion-bypass DNA polymerases in E. coli.
The potent mutagen/carcinogen benzo[a]pyrene (B[a]P) is metabolically activated to (+)-anti-B[a]PDE, which induces a full spectrum of mutations (e.g., G-to-T, G-to-A, -1 frameshifts, etc.) via its major adduct [+ta]-B[a]P-N2-dG. We recently showed that the dominant G-to-T mutation depends on DNA polymerase V (DNAP V), but not DNAPs IV or II, when studied in a 5'-TG sequence in E. coli. Herein we investigate what DNAPs are responsible for non-mutagenic bypass with [+ta]-B[a]P-N2-dG, along with its mirror image adduct [-ta]-B[a]P-N2-dG. Each adduct is built into a 5'-TG sequence in a single stranded M13 phage vector, which is then transformed into eight different E. coli strains containing all combinations of proficiency and deficiency in the three lesion-bypass DNAPs II, IV and V. Based on M13 progeny output, non-mutagenic bypass with [-ta]-B[a]P-N2-dG depends on DNAP IV. In contrast, non-mutagenic bypass with [+ta]-B[a]P-N2-dG depends on both DNAPs IV and V, where arguments suggest that DNAP IV is involved in dCTP insertion, while DNAP V is involved in extension of the adduct-G:C base pair. Numerous findings indicate that DNAP II has a slight inhibitory effect on the bypass of [+ta]- and [-ta]-B[a]P-N2-dG in the case of both DNAPs IV and V. In conclusion, for efficient non-mutagenic bypass (dCTP insertion) in E. coli, [+ta]-B[a]P-N2-dG requires DNAPs IV and V, [-ta]-B[a]P-N2-dG requires only DNAP IV, while DNAP II is inhibitory to both, and experiments to investigate these differences should provide insights into the mechanism and purpose of these lesion-bypass DNAPs.
Seo
Kwang Young
KY
Biology Department, Boston University, 5 Cummington Street, Boston, MA 02215, USA.
Nagalingam
Arumugam
A
Miri
Shadi
S
Yin
Jun
J
Chandani
Sushil
S
Kolbanovskiy
Alexander
A
Shastry
Anant
A
Loechler
Edward L
EL
D017104:Bacteriophage M13 / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000737:chemistry*; D018736:DNA Adducts / Q000737:chemistry*; D004269:DNA, Bacterial / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D009690:Nucleic Acid Conformation; D013237:Stereoisomerism
D018736:DNA Adducts; D004269:DNA, Bacterial; D001564:Benzo(a)pyrene; D004259:DNA-Directed DNA Polymerase