Translesion synthesis across polycyclic aromatic hydrocarbon diol epoxide adducts of deoxyadenosine by Sulfolobus solfataricus DNA polymerase Dpo4

Chem Res Toxicol. 2006 Jun;19(6):859-67. doi: 10.1021/tx060056s.

Abstract

The mechanisms by which derivatives of polycyclic aromatic hydrocarbons (PAHs) cause mutations have been of considerable interest. Three different N(6)-adenyl PAH-diol epoxide oligonucleotide derivatives were studied with the archebacterial translesion DNA polymerase Sulfolobus solfataricus Dpo4. Steady-state kinetic analysis indicated insertion of all four dNTPs opposite each of the three N(6)-adenyl PAH adducts, with only slightly varying misincorporation efficiencies. Full-length extension of shorter primers paired with templates containing the N(6)-adenyl PAH derivatives proceeded to apparent completion at 45 degrees C in the presence of added dimethyl sulfoxide. Analysis of the products by high-performance liquid chromatography/collision-induced mass spectrometry indicated the presence of mixtures of products with each PAH adduct. These mixtures correspond to both error-free synthesis and mixtures of polymerization/realignment steps. With an unmodified template, only the expected A:T and G:C pairing was detected in the primer extension products under these conditions, with no frameshifts. These results demonstrate the complexity of polymerization opposite these bulky N(6)-adenyl PAH adducts, even with a single polymerase.

Publication types

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

MeSH terms

  • DNA-Directed DNA Polymerase / metabolism*
  • Deoxyadenosines / chemistry*
  • Deoxyadenosines / metabolism*
  • Deoxyribonucleotides / metabolism
  • Epoxy Compounds / chemistry*
  • Epoxy Compounds / metabolism*
  • Gas Chromatography-Mass Spectrometry
  • Kinetics
  • Molecular Structure
  • Oligonucleotides / metabolism
  • Polycyclic Aromatic Hydrocarbons / chemistry*
  • Polycyclic Aromatic Hydrocarbons / metabolism*
  • Sulfolobus solfataricus / enzymology*
  • Temperature

Substances

  • Deoxyadenosines
  • Deoxyribonucleotides
  • Epoxy Compounds
  • Oligonucleotides
  • Polycyclic Aromatic Hydrocarbons
  • DNA-Directed DNA Polymerase