Mechanistic studies of the bypass of a bulky single-base lesion catalyzed by a Y-family DNA polymerase

J Biol Chem. 2009 Mar 6;284(10):6379-88. doi: 10.1074/jbc.M808161200. Epub 2009 Jan 5.


1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dGAP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.

Publication types

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

MeSH terms

  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Catalysis
  • DNA Adducts / chemistry*
  • DNA Adducts / genetics
  • DNA Adducts / metabolism
  • DNA Damage / physiology
  • DNA, Archaeal / chemistry*
  • DNA, Archaeal / genetics
  • DNA, Archaeal / metabolism
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • Kinetics
  • Mutation
  • Pyrenes / chemistry*
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / genetics
  • Vehicle Emissions


  • Archaeal Proteins
  • DNA Adducts
  • DNA, Archaeal
  • Pyrenes
  • Vehicle Emissions
  • DNA-Directed DNA Polymerase
  • 1-nitropyrene