Structural Insights Into Complete Metal Ion Coordination From Ternary Complexes of B Family RB69 DNA Polymerase

Biochemistry. 2011 Oct 25;50(42):9114-24. doi: 10.1021/bi201260h. Epub 2011 Sep 29.

Abstract

We have captured a preinsertion ternary complex of RB69 DNA polymerase (RB69pol) containing the 3' hydroxyl group at the terminus of an extendable primer (ptO3') and a nonhydrolyzable 2'-deoxyuridine 5'-α,β-substituted triphosphate, dUpXpp, where X is either NH or CH(2), opposite a complementary templating dA nucleotide residue. Here we report four structures of these complexes formed by three different RB69pol variants with catalytically inert Ca(2+) and four other structures with catalytically competent Mn(2+) or Mg(2+). These structures provide new insights into why the complete divalent metal-ion coordination complexes at the A and B sites are required for nucleotidyl transfer. They show that the metal ion in the A site brings ptO3' close to the α-phosphorus atom (Pα) of the incoming dNTP to enable phosphodiester bond formation through simultaneous coordination of both ptO3' and the nonbridging Sp oxygen of the dNTP's α-phosphate. The coordination bond length of metal ion A as well as its ionic radius determines how close ptO3' can approach Pα. These variables are expected to affect the rate of bond formation. The metal ion in the B site brings the pyrophosphate product close enough to Pα to enable pyrophosphorolysis and assist in the departure of the pyrophosphate. In these dUpXpp-containing complexes, ptO3' occupies the vertex of a distorted metal ion A coordination octahedron. When ptO3' is placed at the vertex of an undistorted, idealized metal ion A octahedron, it is within bond formation distance to Pα. This geometric relationship appears to be conserved among DNA polymerases of known structure.

Publication types

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

MeSH terms

  • Amino Acid Substitution / genetics
  • Calcium / chemistry*
  • Crystallography, X-Ray
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / classification
  • DNA-Directed DNA Polymerase / genetics
  • Diphosphates / chemistry
  • Hydrogen Bonding
  • Magnesium / chemistry*
  • Manganese / chemistry*
  • Nucleic Acid Conformation
  • Nucleic Acid Heteroduplexes / chemistry
  • Phosphorus / chemistry*
  • Viral Proteins / chemistry*
  • Viral Proteins / classification
  • Viral Proteins / genetics

Substances

  • Diphosphates
  • Nucleic Acid Heteroduplexes
  • Viral Proteins
  • Phosphorus
  • Manganese
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase
  • Magnesium
  • Calcium

Associated data

  • PDB/3NCI
  • PDB/3S9H
  • PDB/3SCX
  • PDB/3SI6
  • PDB/3SJJ
  • PDB/3SNN
  • PDB/3SPY
  • PDB/3SPZ
  • PDB/3SQ0
  • PDB/3SQ1