| The well-studied aromatic amine carcinogen, N-2-acetylaminofluorene (AAF), forms adducts at the C8 position of guanine in DNA. Unlike replicative polymerases, Y-family polymerases have been shown to have the ability to bypass such bulky DNA lesions. To better understand the mechanism of translesion synthesis by the yeast DNA polymerase eta (yPoleta), a gel retardation technique was used to measure equilibrium dissociation constants of this polymerase to unmodified DNA or DNA containing dG-C8-AAF or the related deacylated dG-C8-AF adduct. These results show that the binding of yPoleta to the unmodified primer-template is substantially stronger in the presence of the next correct nucleotide than when no or an incorrect nucleotide is present. In addition, binding of yPoleta to either AAF or AF-modified templates is also stronger in the presence of dCTP. Finally, the yPoleta complex is destabilized if the primer extends to a position across from the adduct and stronger binding is not observed in the presence of the next correct nucleotide. Taken together, these data suggest that the yPoleta can undergo a conformational change to a closed ternary complex when an AAF or AF adduct is present in the active site in the presence of the next correct nucleotide and that dissociation is promoted once incorporation occurs.;The similar experiments with human DNA polymerase eta (hPol eta) showed that there is a stronger binding with an unmodified primer template the when correct nucleotide is present compared to no nucleotide. However, binding of hPol eta to an AAF/AF-modified primer template or a template containing an abasic site showed negligible change in it's affinity when either correct or incorrect dNTPs are present.;Binding assays with yPoleta and DNA containing TT dimer showed that yPoleta binds stronger when dATP is present opposite both positions of the TT dimer compared to when no nucleotide present. These results suggest a conformational change to closed complex, similar to what was observed with AAF or AF-modified primer templates. |
