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To analyze translesion DNA synthesis (TLS) across lesions derived from the air pollutant 3-nitrobenzanthrone in Escherichia coli , we constructed site-specifically modified plasmids containing single molecule adducts derived from 3-nitrobenzanthrone. For this experiment, we adopted a modified version of the method developed by Fuchs et al. [29] . Each plasmid contained one of the following lesions in its LacZ ′ gene: N -(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8- N -ABA); 2-(2′-deoxyguanosin- N 2 -yl)-3-aminobenzanthrone (dG- N 2 -C2-ABA); 2-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-C2-ABA); 2-(2′-deoxyadenosin- N 6 -yl)-3-aminobenzanthrone (dA- N 6 -C2-ABA); N -(2′-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8- N -AcABA); or 2-(2′-deoxyguanosin-8-yl)-3-acetylaminobenzanthrone (dG-C8-C2-AcABA). All of the adducts inhibited DNA synthesis by replicative DNA polymerases in E. coli ; however, the extent of the inhibition varied among the adducts. All five dG-adducts strongly blocked replication by replicative DNA polymerases; however, the dA-adduct only weakly blocked DNA replication. The induction of the SOS response increased the frequency of TLS, which was higher for the dG-C8-C2-ABA, dG-C8- N -AcABA and dG-C8-C2-AcABA adducts than for the other adducts. In our previous study, dG-C8- N -ABA blocked DNA replication more strongly and induced mutations more frequently than dG- N 2 -C2-ABA in human cells. In contrast, in E. coli the frequency of TLS over dG- N 2 -C2-ABA was markedly reduced, even under the SOS + conditions, and dG- N 2 -C2-ABA induced G to T mutations. All of the other adducts were bypassed in a less mutagenic manner. In addition, using E. coli strains that lacked particular DNA polymerases we found that DNA polymerase V was responsible for TLS over dG-C8- N -AcABA and dG-C8-C2-AcABA adducts.