Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3'-next flanking base (G*CT vs G*CA; G*, FABP, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl; FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair ( Jain , V. , Hilton , B. , Lin , B. , Patnaik , S. , Liang , F. , Darian , E. , Zou , Y. , Mackerell , A. D. , Jr. , and Cho , B. P. ( 2013 ) Nucleic Acids Res. , 41 , 869 - 880 ). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo(-)) and Dpo4. We obtained dynamic (19)F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5'-CTTACCATCG*CAACCATTC-3')] and G*CT [d(5'-CTTACCATCG*CTACCATTC-3')]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct's chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo(-) vs Dpo4). Steady-state kinetics analysis with Kf-exo(-) revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.