Molecular and structural characterization of oxidized ribonucleotide insertion into DNA by human DNA polymerase β

J Biol Chem. 2020 Feb 7;295(6):1613-1622. doi: 10.1074/jbc.RA119.011569. Epub 2019 Dec 31.

Abstract

During oxidative stress, inflammation, or environmental exposure, ribo- and deoxyribonucleotides are oxidatively modified. 8-Oxo-7,8-dihydro-2'-guanosine (8-oxo-G) is a common oxidized nucleobase whose deoxyribonucleotide form, 8-oxo-dGTP, has been widely studied and demonstrated to be a mutagenic substrate for DNA polymerases. Guanine ribonucleotides are analogously oxidized to r8-oxo-GTP, which can constitute up to 5% of the rGTP pool. Because ribonucleotides are commonly misinserted into DNA, and 8-oxo-G causes replication errors, we were motivated to investigate how the oxidized ribonucleotide is utilized by DNA polymerases. To do this, here we employed human DNA polymerase β (pol β) and characterized r8-oxo-GTP insertion with DNA substrates containing either a templating cytosine (nonmutagenic) or adenine (mutagenic). Our results show that pol β has a diminished catalytic efficiency for r8-oxo-GTP compared with canonical deoxyribonucleotides but that r8-oxo-GTP is inserted mutagenically at a rate similar to those of other common DNA replication errors (i.e. ribonucleotide and mismatch insertions). Using FRET assays to monitor conformational changes of pol β with r8-oxo-GTP, we demonstrate impaired pol β closure that correlates with a reduced insertion efficiency. X-ray crystallographic analyses revealed that, similar to 8-oxo-dGTP, r8-oxo-GTP adopts an anti conformation opposite a templating cytosine and a syn conformation opposite adenine. However, unlike 8-oxo-dGTP, r8-oxo-GTP did not form a planar base pair with either templating base. These results suggest that r8-oxo-GTP is a potential mutagenic substrate for DNA polymerases and provide structural insights into how r8-oxo-GTP is processed by DNA polymerases.

Keywords: 8-oxoguanine (8-oxo-G); DNA damage; DNA polymerase; DNA repair; DNA replication; mutagenic nucleobase; nucleotidyl transferase reaction; oxidized ribonucleotide; r8-oxo-G lesion; structural biology.

Publication types

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

MeSH terms

  • DNA / chemistry
  • DNA / metabolism*
  • DNA Polymerase beta / metabolism*
  • Deoxyguanine Nucleotides / chemistry
  • Deoxyguanine Nucleotides / metabolism*
  • Humans
  • Molecular Docking Simulation
  • Oxidation-Reduction
  • Oxidative Stress
  • Ribonucleotides / chemistry
  • Ribonucleotides / metabolism*

Substances

  • Deoxyguanine Nucleotides
  • Ribonucleotides
  • 8-oxodeoxyguanosine triphosphate
  • DNA
  • DNA Polymerase beta
  • POLB protein, human

Associated data

  • PDB/4UB4
  • PDB/3C2M
  • PDB/4UAW
  • PDB/2FMS
  • PDB/6UOK