Structural mechanism of ribonucleotide discrimination by a Y-family DNA polymerase

J Mol Biol. 2011 Apr 1;407(3):382-90. doi: 10.1016/j.jmb.2011.01.037. Epub 2011 Feb 3.


The ability of DNA polymerases to differentiate between ribonucleotides and deoxribonucleotides is fundamental to the accurate replication and maintenance of an organism's genome. The active sites of Y-family DNA polymerases are highly solvent accessible, yet these enzymes still maintain a high selectivity towards deoxyribonucleotides. Here, we biochemically demonstrate that a single active-site mutation (Y12A) in Dpo4, a model Y-family DNA polymerase, causes both a dramatic loss of ribonucleotide discrimination and a decrease in nucleotide incorporation efficiency. We also determined two ternary crystal structures of the Dpo4 Y12A mutant incorporating either dATP or ATP nucleotides opposite a template dT base. Interestingly, both dATP and ATP were hydrolyzed to dADP and ADP, respectively. In addition, the dADP and ADP molecules adopt a similar conformation and position at the polymerase active site to a ddADP molecule in the ternary crystal structure of wild-type Dpo4. The Y12A mutant loses stacking interactions with the deoxyribose of dNTP, which destabilizes the binding of incoming nucleotides. The mutation also opens a space to accommodate the 2'-OH group of the ribose of NTP in the polymerase active site. The structural change leads to the reduction in deoxynucleotide incorporation efficiency and allows ribonucleotide incorporation.

Publication types

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

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleotides / chemistry*
  • Deoxyribonucleotides / metabolism
  • Kinetics
  • Models, Molecular
  • Mutation
  • Protein Conformation
  • Ribonucleotides / chemistry*
  • Ribonucleotides / metabolism


  • Deoxyribonucleotides
  • Ribonucleotides
  • DNA-Directed DNA Polymerase