The oxidized nucleotide, 8-oxo-7,8-dihydro-2΄-deoxyguanosine (8-oxoG), is one of the most abundant DNA lesions. 8-oxoG plays a major role in tumorigenesis and human disease. Biological consequences of 8-oxoG are mediated in part by its insertion into the genome, making it essential to understand how DNA polymerases handle 8-oxoG. Insertion of 8-oxoG is mutagenic when opposite adenine but not when opposite cytosine. However, either result leads to DNA damage at the primer terminus (3΄-end) during the succeeding insertion event. Extension from DNA damage at primer termini remains poorly understood. Using kinetics and time-lapse crystallography, we evaluated how a model DNA polymerase, human polymerase β, accommodates 8-oxoG at the primer terminus opposite cytosine and adenine. Notably, extension from the mutagenic base pair is favored over the non-mutagenic base pair. When 8-oxoG is at the primer terminus opposite cytosine, DNA centric changes lead to a clash between O8 of 8-oxoG and the phosphate backbone. Changes in the extension reaction resulting from the altered active site provide evidence for a stabilizing interaction between Arg254 and Asp256 that serves an important role during DNA synthesis reactions. These results provide novel insights into the impact of damage at the primer terminus on genomic stability and DNA synthesis.

N6-(2-Deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido pyrimidine (Fapy•dG) is a prevalent form of genomic DNA damage. Fapy•dG is formed in greater amounts under anoxic conditions than the well-studied, chemically related 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodGuo). Fapy•dG is more mutagenic in mammalian cells than 8-oxodGuo. A distinctive property of Fapy•dG is facile epimerization, but prior works with Fapy•dG analogues have precluded determining its effect on chemistry. We present crystallographic characterization of natural Fapy•dG in duplex DNA and as the template base for DNA polymerase β (Pol β). Fapy•dG adopts the β-anomer when base paired with cytosine but exists as a mixture of αand β-anomers when promutagenically base paired with adenine. Rotation about the bond between the glycosidic nitrogen atom and the pyrimidine ring is also affected by the opposing nucleotide. Sodium cyanoborohydride soaking experiments trap the ring-opened Fapy•dG, demonstrating that ring opening and epimerization occur in the crystalline state. Ring opening and epimerization are facilitated by propitious water molecules that are observed in the structures. Determination of Fapy•dG mutagenicity in wild type and Pol β knockdown HEK 293T cells indicates that Pol β contributes to G → T transversions but also suppresses G → A transitions. Complementary kinetic studies have determined that Fapy•dG promotes mutagenesis by decreasing the catalytic efficiency of dCMP insertion opposite Fapy•dG, thus reducing polymerase fidelity. Kinetic studies have determined that dCMP incorporation opposite the β-anomer is ∼90 times faster than the α-anomer. This research identifies the importance of anomer dynamics, a feature unique to formamidopyrimidines, when considering the incorporation of nucleotides opposite Fapy•dG and potentially the repair of this structurally unusual lesion.

DNA polymerases are vital for the synthesis of new DNA strands. Since the discovery of DNA polymerase I in Escherichia coli, a diverse library of mammalian DNA polymerases involved in DNA replication, DNA repair, antibody generation, and cell checkpoint signaling has emerged. While the unique functions of these DNA polymerases are differentiated by their association with accessory factors and/or the presence of distinctive catalytic domains, atomic resolution structures of DNA polymerases in complex with their DNA substrates have revealed mechanistic subtleties that contribute to their specialization. In this review, the structure and function of all 15 mammalian DNA polymerases from families B, Y, X, and A will be reviewed and discussed with special emphasis on the insights gleaned from recently published atomic resolution structures.

4,6-Diamino-5-formamidopyrimidine (Fapy•dG) is an abundant form of oxidative DNA damage that is mutagenic and contributes to the pathogenesis of human disease. When Fapy•dG is in its nucleotide triphosphate form, Fapy•dGTP, it is inefficiently cleansed from the nucleotide pool by the responsible enzyme in Escherichia coli MutT and its mammalian homolog MTH1. Therefore, under oxidative stress conditions, Fapy•dGTP could become a pro-mutagenic substrate for insertion into the genome by DNA polymerases. Here, we evaluated insertion kinetics and high-resolution ternary complex crystal structures of a configurationally stable Fapy•dGTP analog, β-C-Fapy•dGTP, with DNA polymerase β. The crystallographic snapshots and kinetic data indicate that binding of β-C-Fapy•dGTP impedes enzyme closure, thus hindering insertion. The structures reveal that an active site residue, Asp276, positions β-C-Fapy•dGTP so that it distorts the geometry of critical catalytic atoms. Removal of this guardian side chain permits enzyme closure and increases the efficiency of β-C-Fapy•dG insertion opposite dC. These results highlight the stringent requirements necessary to achieve a closed DNA polymerase active site poised for efficient nucleotide incorporation and illustrate how DNA polymerase β has evolved to hinder Fapy•dGTP insertion.