Bidentate and tridentate metal-ion coordination states within ternary complexes of RB69 DNA polymerase

Protein Sci. 2012 Mar;21(3):447-51. doi: 10.1002/pro.2026. Epub 2012 Jan 31.

Abstract

Two divalent metal ions are required for primer-extension catalyzed by DNA polymerases. One metal ion brings the 3'-hydroxyl of the primer terminus and the α-phosphorus atom of incoming dNTP together for bond formation so that the catalytically relevant conformation of the triphosphate tail of the dNTP is in an α,β,γ-tridentate coordination complex with the second metal ion required for proper substrate alignment. A probable base selectivity mechanism derived from structural studies on Dpo4 suggests that the inability of mispaired dNTPs to form a substrate-aligned, tridentate coordination complex could effectively cause the mispaired dNTPs to be rejected before catalysis. Nevertheless, we found that mispaired dNTPs can actually form a properly aligned tridentate coordination complex. However, complementary dNTPs occasionally form misaligned complexes with mutant RB69 DNA polymerases (RB69pols) that are not in a tridentate coordination state. Here, we report finding a β,γ-bidentate coordination complex that contained the complementary dUpNpp opposite dA in the structure of a ternary complex formed by the wild type RB69pol at 1.88 Å resolution. Our observations suggest that several distinct metal-ion coordination states can exist at the ground state in the polymerase active site and that base selectivity is unlikely to be based on metal-ion coordination alone.

Publication types

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

MeSH terms

  • Calcium / chemistry
  • Cations, Divalent / chemistry*
  • Crystallography, X-Ray
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleotides / metabolism
  • Magnesium / chemistry
  • Metals / chemistry*
  • Models, Molecular
  • Molecular Conformation
  • Protein Conformation
  • Viral Proteins / chemistry*

Substances

  • Cations, Divalent
  • Deoxyribonucleotides
  • Metals
  • Viral Proteins
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase
  • Magnesium
  • Calcium

Associated data

  • PDB/3UIQ