A deoxyinosine specific endonuclease from hyperthermophile, Archaeoglobus fulgidus: a homolog of Escherichia coli endonuclease V.
Deoxyadenosine undergoes spontaneous deamination to deoxyinosine in DNA. Based on amino acids sequence homology, putative homologs of endonuclease V were identified in several organisms including archaebacteria, eubacteria as well as eukaryotes. The translated amino acid sequence of the Archaeoglobus fulgidus nfi gene shows 39% identity and 55% similarity to the E. coli nfi 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 was purified to apparent homogeneity by a combination of Ni(++) affinity and MonoS cation exchange liquid chromatography. The purified C-terminal fusion protein has a molecular weight of about 25kDa and showed endonuclease activity towards DNA containing deoxyinosine. A. fulgidus endonuclease V has an absolute requirement for Mg(2+) and an optimum reaction temperature at 85 degrees C. However, in contrast to E. coli endonuclease V, which has a wide substrate spectrum, endonuclease V from A. fulgidus recognized only deoxyinosine. These data suggest that the deoxyinosine cleavage activity is a primordial activity of endonuclease V and that multiple enzymatic activities of E. coli endonuclease V were acquired later during evolution.
Liu
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Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Ave, Atlanta, GA 30335, USA.
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Qing
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D000595:Amino Acid Sequence; D019609:Archaeoglobus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004269:DNA, Bacterial; D007288:Inosine; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C012271:deoxyinosine