Unexpected Behavior of DNA Polymerase Mu Opposite Template 8-oxo-7,8-dihydro-2'-guanosine

Nucleic Acids Res. 2019 Sep 26;47(17):9410-9422. doi: 10.1093/nar/gkz680.

Abstract

DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase μ (Pol μ) in steady-state kinetics and cell-based assays. Pol μ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol μ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol μ active site with none of the DNA substrate distortions observed for Family X siblings Pols β or λ. Kinetic characterization of template 8OG bypass indicates that Pol μ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / genetics
  • DNA Breaks, Double-Stranded / radiation effects*
  • DNA Damage / genetics
  • DNA Damage / radiation effects
  • DNA End-Joining Repair / genetics*
  • DNA End-Joining Repair / radiation effects
  • DNA Ligase ATP / genetics
  • DNA Replication / genetics
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / genetics*
  • Guanosine / analogs & derivatives*
  • Guanosine / genetics
  • Humans
  • Mutagenesis / radiation effects
  • Radiation, Ionizing
  • Reactive Oxygen Species / chemistry
  • Ultraviolet Rays

Substances

  • Reactive Oxygen Species
  • Guanosine
  • 8-hydroxyguanosine
  • Adenosine Triphosphate
  • DNA polymerase mu
  • DNA-Directed DNA Polymerase
  • DNA Ligase ATP