| Popis: |
DNA polymerases must accurately replicate DNA to maintain the integrity of the genome. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), are covalently bound to bases on DNA leading to altered DNA polymerase activity and increased levels of mutagenesis at the adduct site. Although AF and AAF adducts differ only by an acetyl group, the two adducts have different biological effects and the AF adduct is much more easily bypassed by most DNA polymerases. While carcinogen-induced mutagenesis has been extensively studied using ensemble experiments, the exact mechanism for how DNA damage leads to a mutation remains unclear. We have used two complementary single-molecule methods, fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement (smPIFE), to characterize the interactions between a DNA polymerase and DNA containing an AF or AAF adduct. These studies have allowed us to gain unprecedented insights into the molecular dynamics occurring between a DNA polymerase and a damaged template. We found the degree to which an adduct destabilizes polymerase binding to the DNA depends on the location of the adduct with respect to the primer terminus, the type of adduct, and the nucleotides present in the solution. Not only do the adducts influence the dwell time of the polymerase on the DNA, but also its binding position and orientation. Finally, we have for the first time directly observed the adduct-induced internal transfer of the DNA from the polymerization active site to the 3’-5’ exonuclease site. |
