Structural and kinetic insights into binding and incorporation of L-nucleotide analogs by a Y-family DNA polymerase

Nucleic Acids Res. 2014 Sep;42(15):9984-95. doi: 10.1093/nar/gku709. Epub 2014 Aug 7.


Considering that all natural nucleotides (D-dNTPs) and the building blocks (D-dNMPs) of DNA chains possess D-stereochemistry, DNA polymerases and reverse transcriptases (RTs) likely possess strongD-stereoselectivity by preferably binding and incorporating D-dNTPs over unnatural L-dNTPs during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by DNA polymerases or RTs has not been established although L-deoxycytidine analogs (lamivudine and emtricitabine) and L-thymidine (telbivudine) have been widely used as antiviral drugs for years. Here we report seven high-resolution ternary crystal structures of a prototype Y-family DNA polymerase, DNA, and D-dCTP, D-dCDP, L-dCDP, or the diphosphates and triphosphates of lamivudine and emtricitabine. These structures reveal that relative to D-dCTP, each of these L-nucleotides has its sugar ring rotated by 180° with an unusual O4'-endo sugar puckering and exhibits multiple triphosphate-binding conformations within the active site of the polymerase. Such rare binding modes significantly decrease the incorporation rates and efficiencies of these L-nucleotides catalyzed by the polymerase.

Publication types

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

MeSH terms

  • Catalytic Domain
  • DNA / chemistry
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / metabolism
  • Deoxycytidine / analogs & derivatives
  • Deoxycytidine / chemistry
  • Deoxycytosine Nucleotides / chemistry*
  • Deoxycytosine Nucleotides / metabolism
  • Drug Resistance, Viral
  • Emtricitabine
  • Kinetics
  • Lamivudine / chemistry
  • Models, Molecular
  • Reverse Transcriptase Inhibitors
  • Stereoisomerism
  • Sulfolobus solfataricus / enzymology


  • Deoxycytosine Nucleotides
  • Reverse Transcriptase Inhibitors
  • Deoxycytidine
  • 2'-deoxycytidine 5'-triphosphate
  • Lamivudine
  • 2'-deoxycytidine diphosphate
  • DNA
  • DNA Polymerase beta
  • Emtricitabine

Associated data

  • PDB/4QW8
  • PDB/4QW9
  • PDB/4QWA
  • PDB/4QWB
  • PDB/4QWC
  • PDB/4QWD
  • PDB/4QWE