Partial purification and characterization of an endonuclease from spinach that cleaves ultraviolet light-damaged duplex DNA.
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.
Doetsch
P W
PW
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
McCray
W H
WH
Valenzuela
M R
MR
D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004492:Edetic Acid / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000302:isolation & purification*; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology; D014466:Ultraviolet Rays; D015032:Zinc / Q000494:pharmacology
D004277:DNA, Single-Stranded; D012965:Sodium Chloride; D004247:DNA; D004492:Edetic Acid; D004706:Endodeoxyribonucleases; D015032:Zinc