Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged...

An endonuclease that cleaves ultraviolet light (UV)-damaged, supercoiled plasmid DNA was partially purified from spinach leaves (Spinacia oleracea) by a series of column chromatography steps. Dialysis of the enzyme against EDTA resulted in a greater than 90% loss of activity which could be fully restored following the addition of Zn2+, suggesting that divalent cations are associated with the active enzyme. The spinach endonuclease cleaved duplex, UV-damaged, end-labelled DNA of defined sequence at positions of adenine in the presence of salt (KH2PO4 or NaCl) concentrations of 50 mM or higher. Cleavage of UV-irradiated DNA was dose-dependent and increased steadily within a fluence range of 50-10,000 J/m2. The UV damage requirement and adenine cleavage specificity could be eliminated with lower salt concentrations (0-25 mM), suggesting that the endonuclease recognizes and incises single-stranded DNA. The properties of this enzyme, which we have termed nuclease SP, suggest that it may mediate a role in DNA repair and/or recombination processes in spinach.