Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase

J Mol Biol. 2007 Jan 19;365(3):590-602. doi: 10.1016/j.jmb.2006.10.008. Epub 2006 Oct 7.


Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.

MeSH terms

  • Binding Sites
  • Catalysis
  • Crystallography, X-Ray
  • DNA / biosynthesis*
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / metabolism*
  • Deoxyadenine Nucleotides / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoretic Mobility Shift Assay
  • Kinetics
  • Models, Molecular
  • Nucleotides / chemistry
  • Nucleotides / metabolism*
  • Protein Structure, Secondary
  • Pyrenes / chemistry
  • Pyrenes / metabolism
  • Substrate Specificity
  • Sulfolobus solfataricus / enzymology*
  • Templates, Genetic*


  • Deoxyadenine Nucleotides
  • Nucleotides
  • Pyrenes
  • pyrene deoxynucleotide
  • DNA
  • DNA Polymerase beta
  • 2'-deoxyadenosine triphosphate

Associated data

  • PDB/2IMW