Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity

J Biol Chem. 2002 Mar 8;277(10):8235-42. doi: 10.1074/jbc.M107286200. Epub 2001 Dec 26.


Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

MeSH terms

  • Arginine / chemistry
  • Aspartic Acid / chemistry
  • Base Pair Mismatch
  • Binding Sites
  • Catalysis
  • DNA / metabolism
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / metabolism*
  • Dose-Response Relationship, Drug
  • Glycine / chemistry
  • Humans
  • Hydrogen Bonding
  • Kinetics
  • Lysine / chemistry
  • Models, Chemical
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Purines / chemistry*
  • Pyrimidines / chemistry*
  • Thymine Nucleotides / metabolism


  • Purines
  • Pyrimidines
  • Thymine Nucleotides
  • Aspartic Acid
  • DNA
  • Arginine
  • DNA Polymerase beta
  • Lysine
  • thymidine 5'-triphosphate
  • Glycine