Distinct Roles of the Active-Site Mg2+ Ligands, Asp882 and Asp705, of DNA Polymerase I (Klenow Fragment) During the Prechemistry Conformational Transitions

J Biol Chem. 2011 Feb 4;286(5):3755-66. doi: 10.1074/jbc.M110.167593. Epub 2010 Nov 16.

Abstract

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.

Publication types

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

MeSH terms

  • Aspartic Acid / physiology*
  • Catalysis
  • Catalytic Domain*
  • DNA Polymerase I / chemistry*
  • Ligands
  • Magnesium / metabolism*
  • Phase Transition
  • Protein Conformation

Substances

  • Ligands
  • Aspartic Acid
  • DNA Polymerase I
  • Magnesium