Structures of DNA polymerases caught processing size-augmented nucleotide probes.

The integrity of the genome relies primarily on the ability of DNA polymerases to efficiently catalyze selective DNA synthesis according to the Watson–Crick rule in a templatedirected manner during DNA replication, repair, and recombination. Remarkably, some DNA polymerases achieve selective information transfer to the offspring in line with the Watson–Crick rule with intrinsic error rates as low as one mistake per one million synthesized nucleotides. This is far below the value that would be expected based on the energetic differences between canonical (i.e. Watson–Crick) and noncanonical nucleobase pairing. Geometric factors are widely cited to govern DNA polymerase selectivity. Thus, high-fidelity DNA polymerases are believed to mostly select the canonical nucleotide based on close steric complementarity of the nascent base pair to the geometry of the active site of the enzyme. Synthetic nucleotide analogues with tailored new properties are widely used to investigate the mechanisms that govern DNA polymerase selectivity. In this context nucleotides with hydrophobic nucleobase isosteres were developed to study the contribution of hydrogen bonding to DNA polymerase selectivity. 3] To investigate the steric effects in interactions between DNA polymerase and its substrate, we have developed 4’-alkyl-modified nucleotides with increasing steric demand. The modifications were introduced in the sugar residues since structural and functional data of DNA polymerases show that the sugar residues of nucleotides are involved in substrate recognition. These interactions may provide the enzymes with additional paths for achieving selectivity besides editing nucleobase geometry. Alkyl groups were employed since their effects on the hydrogen-bonding patterns and conformations of the nucleotides would be minimized. By employing these size-augmented nucleotides in functional DNA polymerase studies we could evaluate varied steric effects on DNA polymerase selectivity. Herein we report the first crystal structures of size-augmented 4’methylated and 4’-ethylated thymidine triphosphates in complex with a DNA polymerase. Significant mechanistic insights into nucleotide incorporation during DNA polymerization were derived from the high-resolution crystal structures reported by Waksman and colleagues of KlenTaq, an N-terminally truncated form of DNA polymerase from Thermus aquaticus. KlenTaq is a member of the family A DNA polymerases that play a role in prokaryotic and eukaryotic DNA replication and repair. Since data on the action of KlenTaq on 4’-alkylated thymidine-5’-triphosphates (dTTPs) is lacking, we first studied nucleotide incorporation by employing transient kinetic analysis with quench–flow technology (Table 1). Our data