Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 115, Issue 40, Pages 9992-9997Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1811518115
Keywords
DNA polymerase; modified nucleotide; crystallography; alkyne; click chemistry
Categories
Funding
- Deutsche Forschungsgemeinschaft (DFG)
- European Research Council (AdG) [339834 EvoEPIGEN]
- Swiss Light Source of the Paul Scherrer Institute
- state of Baden-Wurttemberg
- DFG [INST 40/467-1 FUGG]
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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 nudeotide 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.
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