Bulky DNA adducts often exist in multiple conformations that are slowly interconverted to one another. Different conformations have shown to lead to different kinetic and binding capacities, presumably resulting in different mutational consequences. Studies on conformation-specific replication block are lacking due to the complex dynamic nature of DNA replication. A bulky lesion-induced replication block is difficult to capture because existing assays usually detect a consequence of all conformers or the culmination of cellular behaviors. Recently, we have mapped all 16-possible DNA duplexes (CTTCTNG*NCCTC: N= A, G, C, T; G*=ABP) containing the human bladder carcinogen 4-aminobiphenyl (ABP). The results revealed a striking sequence-dependent mixture of B-type (B)/stacked (S) conformations (TG1*G2T [67%B:33%S] and TG1G2*T [100%B]). In this study, we attempted to correlate replication block to different conformations derived from the bulky ABP lesion. We utilized a combination of surface plasmon resonance (SPR) and steady-state kinetics as well as E. coli in-cell replication bypass assays to study the conformational differences in vitro and in the cell. The SPR results indicated that the bulky ABP lesion increases polymerase binding affinity and decreases nucleotide selectivity. The Lineweaver–Burke enzyme inhibition model suggested that the S-conformer may play as a replication blocker.

Conformation-specific replication block from bulky 4-aminobiphenyl-modified DNA lesions

256th National Meeting (2018)

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