Building better polymerases: Engineering the replication of expanded genetic alphabets

J Biol Chem. 2020 Dec 11;295(50):17046-17059. doi: 10.1074/jbc.REV120.013745. Epub 2020 Oct 1.


DNA polymerases are today used throughout scientific research, biotechnology, and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for science-guided rational (or semi-rational) engineering to identify variants that replicate unnatural base pairs (UBPs), unnatural backbones, tags, or other evolutionarily novel features of unnatural DNA. In this review, we provide a brief overview of the chemistry and properties of replicative DNA polymerases and their evolved variants, focusing on the Klenow fragment of Taq DNA polymerase (Klentaq). We describe comparative structural, enzymatic, and molecular dynamics studies of WT and Klentaq variants, complexed with natural or noncanonical substrates. Combining these methods provides insight into how specific amino acid substitutions distant from the active site in a Klentaq DNA polymerase variant (ZP Klentaq) contribute to its ability to replicate UBPs with improved efficiency compared with Klentaq. This approach can therefore serve to guide any future rational engineering of replicative DNA polymerases.

Keywords: DNA polymerase; X-ray crystallography; biotechnology; computer modeling; directed evolution; enzyme mechanism; enzyme structure; expanded genetic alphabets; molecular dynamics; nucleoside/nucleotide analogue; protein-DNA interaction; synthetic biology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Review

MeSH terms

  • DNA / biosynthesis*
  • DNA / genetics
  • DNA Replication*
  • Protein Engineering*
  • Taq Polymerase* / chemistry
  • Taq Polymerase* / genetics


  • DNA
  • Taq Polymerase

Associated data

  • PDB/5W6Q
  • PDB/5W6K
  • PDB/5DKU
  • PDB/4DQS
  • PDB/1KFD
  • PDB/2BPD
  • PDB/4XVI
  • PDB/6VDE
  • PDB/3RTV
  • PDB/3SZ2
  • PDB/3KTQ
  • PDB/4KTQ
  • PDB/4DQQ