Snapshots of a modified nucleotide moving through the confines of a DNA polymerase

Significance Despite being evolved to process the four canonical nucleotides, DNA polymerases are known to incorporate and extend from modified nucleotides, which is the key to numerous core biotechnology applications. The structural basis for postincorporation elongation remained elusive. We successfully crystallized KlenTaq DNA polymerase in six complexes, providing high-resolution snapshots of the modification “moving” from the 3′ terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity. This highlights the unexpected plasticity of the system as origin of the broad substrate properties of the DNA polymerase and guide for the design of improved systems.
DNA polymerases have evolved to process the four canonical nucleotides accurately. Nevertheless, these enzymes are also known to process modified nucleotides, which is the key to numerous core biotechnology applications. Processing of modified nucleotides includes incorporation of the modified nucleotide and postincorporation elongation to proceed with the synthesis of the nascent DNA strand. The structural basis for postincorporation elongation is currently unknown. We addressed this issue and successfully crystallized KlenTaq DNA polymerase in six closed ternary complexes containing the enzyme, the modified DNA substrate, and the incoming nucleotide. Each structure shows a high-resolution snapshot of the elongation of a modified primer, where the modification “moves” from the 3′-primer terminus upstream to the sixth nucleotide in the primer strand. Combining these data with quantum mechanics/molecular mechanics calculations and biochemical studies elucidates how the enzyme and the modified substrate mutually modulate their conformations without compromising the enzyme’s activity significantly. The study highlights the plasticity of the system as origin of the broad substrate properties of DNA polymerases and facilitates the design of improved systems.