Single-Molecule Investigation of Response to Oxidative DNA Damage by a Y-Family DNA Polymerase

Biochemistry. 2016 Apr 12;55(14):2187-96. doi: 10.1021/acs.biochem.6b00166. Epub 2016 Mar 30.

Abstract

Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium toward a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG.

Publication types

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

MeSH terms

  • 8-Hydroxy-2'-Deoxyguanosine
  • Amino Acid Substitution
  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism*
  • Computer Simulation
  • DNA Damage*
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / metabolism*
  • DNA Repair*
  • Deoxyguanosine / analogs & derivatives
  • Deoxyguanosine / chemistry
  • Deoxyguanosine / metabolism
  • Fluorescence Resonance Energy Transfer
  • Models, Molecular*
  • Mutation
  • Oxidation-Reduction
  • Protein Conformation
  • Protein Engineering*
  • Protein Refolding
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins
  • Sulfolobus solfataricus / enzymology*

Substances

  • Archaeal Proteins
  • Recombinant Proteins
  • 8-Hydroxy-2'-Deoxyguanosine
  • DNA Polymerase beta
  • Deoxyguanosine