Noncatalytic aspartate at the exonuclease domain of proofreading DNA polymerases regulates both degradative and synthetic activities

Proc Natl Acad Sci U S A. 2018 Mar 27;115(13):E2921-E2929. doi: 10.1073/pnas.1718787115. Epub 2018 Mar 12.


Most replicative DNA polymerases (DNAPs) are endowed with a 3'-5' exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp121 of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3'-5' exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp90 in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.

Keywords: 3′-5′ exonuclease; DNA polymerase; site-directed mutagenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aspartic Acid / genetics*
  • Bacillus Phages / enzymology*
  • Bacillus Phages / genetics
  • DNA Repair*
  • DNA Replication*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Mutagenesis, Site-Directed
  • Mutation*
  • Sequence Homology


  • Aspartic Acid
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases