Molecular insights into DNA polymerase deterrents for ribonucleotide insertion

J Biol Chem. 2011 Sep 9;286(36):31650-60. doi: 10.1074/jbc.M111.253401. Epub 2011 Jul 6.


DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase β discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn(2+) for Mg(2+) decreased sugar discrimination for both wild-type and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A- and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2' of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (∼2.5 and 2.6 Å, respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ∼2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase β utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Substitution
  • Catalytic Domain
  • Crystallization
  • Crystallography, X-Ray
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / metabolism*
  • Humans
  • Hydrogen Bonding
  • Protein Structure, Tertiary
  • Ribonucleotides / chemistry
  • Ribonucleotides / metabolism*
  • Substrate Specificity
  • Thermodynamics


  • Ribonucleotides
  • DNA Polymerase beta

Associated data

  • PDB/3RH4
  • PDB/3RH5
  • PDB/3RH6