Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

DNA Repair (Amst). 2015 May:29:65-73. doi: 10.1016/j.dnarep.2015.01.012. Epub 2015 Feb 4.


Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2'-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.

Keywords: DNA replication; Enzyme specificity; Hydrogen exchange mass spectrometry; Nucleotide selection; Ribonucleotide.

Publication types

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

MeSH terms

  • Catalytic Domain
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleotides / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Humans
  • Mutation
  • Ribonucleotides / metabolism
  • Substrate Specificity


  • Deoxyribonucleotides
  • DinB protein, E coli
  • Escherichia coli Proteins
  • Ribonucleotides
  • DNA-Directed DNA Polymerase
  • POLK protein, human