Primer extension studies have shown that the Y-family DNA polymerase Dpo4 can bypass the dG-AAF adduct with preferential incorporation of normal partner C, followed by mismatched A [Boudsocq et al. (2001) Nucleic Acids Res. 29:4607]. This polymerase contains major and minor groove pockets that may accommodate a bulky adduct. The major groove pocket is much more spacious and open. Decades of studies have indicated that dG-AAF favors the syn glycosidic torsion conformation. However, this conformation precludes Watson-Crick base-pairing in B-DNA, presenting a challenge to the polymerase in selection of the complementary incoming nucleotide, and in accommodating a C8 substituted bulky adduct which then would be placed in the small minor groove pocket. Our goal in the current study is to elucidate on a structural level how dG-AAF can be harbored in the Dpo4 DNA polymerase active site opposite an incoming dCTP or dATP, using molecular modeling and molecular dynamics simulations. Both anti and syn conformations of the templating dG-AAF in Dpo4 were investigated, employing a Type I Dpo4 ternary complex crystal structure [Ling et al., (2004) Proc. Natl. Acad. Sci.U.S.A. 101:2265] as initial model. We found that a structure with unusual anti glycosidic torsion and C1’-exo deoxyribose conformation allows dG-AAF to be Watson-Crick hydrogen-bonded with dCTP, or form two hydrogen bonds with an incoming syn dATP. In these structures, the fluorenyl rings of dG-AAF are comfortably situated in the Dpo4 large major groove open pocket. With dG-AAF syn, the fluorenyl rings are in the small minor groove pocket and the active site region is highly distorted. Thus, flexibilities inherent in DNA structure can be utilized by the Dpo4 DNA polymerase to facilitate bypass of a bulky adduct. The novel anti AAF-dG conformation can explain the preferential incorporation of dC and dA by Dpo4, and also may be relevant to observed dCTP incorporation opposite AAF-dG in other polymerases. This work is supported by NIH 2R01 CA75449.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]