DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine

Abstract

Conditions which result in DNA strand breakage by the rat liver DNA nicking-closing enzyme lead to the covalent attachment of the 3'-end of the broken strand to the enzyme. Treatment of this complex with pancreatic DNase leaves a residue of 17 +/- 8 nucleotide phosphates still attached to the enzyme. Subsequent nuclease P1 treatment removes all but 2 +/- 1 phosphate residues. Using nuclease P1-treated complexes which had been labeled in the DNA with 32P, the stability of the protein-DNA linkage was studied. The linkage is stable to acid, base, neutral and acidic hydroxylamine, and neutral I2. This pattern of stability rules out essentially all of the possible DNA-protein linkages except for a linkage involving a phosphodiester bond to the amino acid tyrosine. After acid hydrolysis of the 32P-labeled complexes, label was found to be associated with O4-phosphotyrosine, providing a direct demonstration that tyrosine is the amino acid to which the end of the DNA chain is attached.