Replication across template T/U by human DNA polymerase-iota

Structure. 2009 Jul 15;17(7):974-80. doi: 10.1016/j.str.2009.04.011.

Abstract

Human DNA polymerase-iota (Poliota) incorporates correct nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines. In fact, the fidelity opposite template T is so poor that Poliota inserts an incorrect dGTP approximately 10 times better than it inserts the correct dATP. We determine here how a template T/U is accommodated in the Poliota active site and why a G is incorporated more efficiently than an A. We show that in the absence of incoming dATP or dGTP (binary complex), template T/U exists in both syn and anti conformations, but in the presence of dATP or dGTP (ternary complexes), template T/U is predominantly in the anti conformation. We also show that dATP and dGTP insert differently opposite template T/U, and that the basis of selection of dGTP over dATP is a hydrogen bond between the N2 amino group of dGTP and Gln59 of Poliota.

Publication types

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

MeSH terms

  • Binding Sites / genetics
  • Crystallography, X-Ray
  • DNA / chemistry
  • DNA / genetics
  • DNA Replication*
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Glutathione Transferase / metabolism
  • Humans
  • Hydrogen Bonding
  • Kinetics
  • Models, Molecular
  • Nucleic Acid Conformation
  • Oligonucleotides / chemical synthesis
  • Oligonucleotides / chemistry
  • Oligonucleotides / isolation & purification
  • Oligonucleotides / metabolism
  • Protein Binding
  • Protein Conformation
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity / genetics
  • Templates, Genetic*
  • Thymine / metabolism*
  • Uracil / metabolism*
  • X-Ray Diffraction

Substances

  • Oligonucleotides
  • Recombinant Fusion Proteins
  • Uracil
  • DNA
  • Glutathione Transferase
  • DNA polymerase iota
  • DNA-Directed DNA Polymerase
  • Thymine

Associated data

  • PDB/3H40
  • PDB/3H4B
  • PDB/3H4D