Biochemical Characterization of Uracil-DNA Glycosylase from Pyrococcus furiosus

We report the characterization of a uracil-DNA glycosylase(UDG) from the hyperthermophilic archaea Pyrococcus furiosus(P. furiosus). P. furiosus UDG(PfUDG) has high sequence similarity to the families IV and V UDGs(thermostable UDG family and PaUDG-b family). PfUDG excises uracil from various DNA substrates with the following order: U/T≈U/C>U/G≈U/AP≈U/->U/U≈U/I≈U/A. The optimal temperature and pH value for uracil excision by PfUDG are 70 ℃ and 9.0, respectively. The removal of U is inhibited by the divalent ions of Fe, Ca, Zn, Cu, Co, Ni and Mn, as well as a high concentration of NaCl. The phosphorothioates near uracil strongly inhibit the excision of uracil by PfUDG. Interestingly, pfuDNA(Pyrococcus furiosus DNA) polymerase, which tightly binds the uracil- carrying oligonucleotide, does not inhibit the excision by PfUDG, suggesting PfUDG in vivo functions as the repair enzyme to excise uracil damage in genome.