A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V

Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Ba sed on amino acids sequence homology, putative homologs of endonuclease V w ere identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobu s fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nf i gene. A. fulgidus endonuclease V was cloned and expressed in E. coli as a C-terminal hexa-histidine fusion protein. The C-terminal fusion protein wa s purified to apparent homogeneity by a combination of Ni++ affinity and Mo noS cation exchange liquid chromatography. The purified C-terminal fusion p rotein has a molecular weight of about 25 kDa and showed endonuclease activ ity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg2+ and an optimum reaction temperature at 85 deg reesC. However, in contrast to E. coli endonuclease V, which has a wide sub strate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosi ne. These data suggest that the deoxyinosine cleavage activity is a primord ial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution. (C) 2000 Elsevie r Science B.V. All rights reserved.