Access Type

Open Access Dissertation

Date of Award

January 2017

Degree Type


Degree Name




First Advisor

Louis J. Romano


DNA is constantly exposed to various DNA damaging agents that are generated by various internal and external sources. Some of this damage may not be able to be repaired by cellular machineries causing DNA replication to be blocked. Once the replication fork is blocked by a DNA adduct, damage tolerance DNA polymerases, mainly Y-family, are able to restore the DNA replication by synthesizing past the DNA adduct. Benzo[a]pyrene (B[a]P) is one of the most studied environmental carcinogens. It is known to make covalent DNA adducts after metabolic activation and the bulkiness of the B[a]P adducts impose a strong barrier to high-fidelity polymerases. However, many Y-family polymerases are able to bypass these adducts. In this study, we used a model Y-family polymerase isolated from Sulfolobus solfataricus called DNA polymerase IV (Dpo4), to study the B[a]P modified DNA bypass mechanism using single molecule FRET (smFRET). The B[a]P reaction with DNA produces four different isomeric products and we selected the most abundant of them, (+)-cis-B[a]P-N2-dG and (+)-trans-B[a]P-N2-dG, to study.

Our data show that (+)-cis-B[a]P-N2-dG adduct blocks the DNA replication when it is in the intercalated conformation in the DNA. Use of DMSO in the reaction buffer supports for the stabilization of the adduct solvent exposed conformation which removes the obstacle in the Dpo4 active site for DNA synthesis. In contrast, (+)-trans-B[a]P-N2-dG adduct does not completely block the DNA replication, but instead causes frame-shift mutation leading to a -1 deletions in the DNA product. Consistence with the previously published studies, Dpo4 moves between two conformations in the binary complex, but single conformation is formed in the ternary complex. However, Dpo4 binding conformations in the normal DNA and modified DNA are clearly different indicating its competence in translesion synthesis.