Effect of N-2-acetylaminofluorene and 2-aminofluorene adducts on DNA binding and synthesis by yeast DNA polymerase eta.

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 for 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 dG-C8-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 are consistent with the ability of yPoleta to undergo a conformational change to a closed ternary complex in the presence of the next correct nucleotide and on templates containing an AAF or AF adduct but do not rule out other possible explanations.