Further characterization of Escherichia coli endonuclease V. Mechanism of recognition for deoxyinosine, deoxyuridine, and base mismatches in DNA.
Endonuclease V from Escherichia coli has a wide substrate spectrum. In addition to deoxyinosine-containing DNA, the enzyme cleaves DNA containing urea residues, AP sites, base mismatches, insertion/deletion mismatches, flaps, and pseudo-Y structures. The gene coding for the enzyme was identified to be orf 225 or nfi (endonuclease five). Using enzyme purified from an overproducing strain, the deoxyinosine- and mismatch-specific activities of endonuclease V was found to have different divalent metal requirements. The affinity of the enzyme is greater than 20-fold higher for DNA containing deoxyinosine than deoxynebularine or base mismatches. Under optimal cleavage conditions, endonuclease V forms two stable complexes with DNA containing deoxyinosine, but not with DNA containing base mismatches or deoxynebularine, suggesting that the 6-keto group of hypoxanthine in DNA is critical for stable interactions with the protein. The enzyme recognizes deoxyuridine in DNA but exhibits a much lower affinity to DNA containing deoxyuridine compared with DNA containing deoxyinosine. Interestingly, deoxyuridine-specific endonuclease activity of endonuclease V has a divalent metal requirement similar to the mismatch activity. A model for the mechanism of substrate recognition is proposed to explain these different activities.
Yao
M
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Division of Cancer Biology, Department of Radiation Oncology Emory University School of Medicine, Atlanta, Georgia 30335, USA.
Kow
Y W
YW
D004247:DNA / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer); D003853:Deoxyribonucleosides / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism; D015636:Magnesium Chloride / Q000378:metabolism; D008970:Molecular Weight; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D011684:Purine Nucleosides / Q000378:metabolism
D003853:Deoxyribonucleosides; D009692:Nucleic Acid Heteroduplexes; D011684:Purine Nucleosides; D015636:Magnesium Chloride; C050393:2'-deoxynebularine; D007288:Inosine; D004247:DNA; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C012271:deoxyinosine; D003857:Deoxyuridine