DEOXYRIBONUCLEIC-ACID KINASE FROM NUCLEI OF RAT-LIVER - MECHANISM, REVERSAL, AND INHIBITORS OF THE REACTION

The DNA kinase from rat liver nuclei has been shown to use ATP to phosphorylate the 5‘-hydroxyl terminus of DNA and longer oligodeoxynucleotides; in contrast to the phage-induced polynucleotide kinase, it is inactive on RNA, low molecular weight oligonucleotides, and nucleoside 3‘-monophosphates (Levin, C. J., & Zimmerman, S. B. (1976) J. Biol. Chem. 251, 1767-1774). Kinetic measurements indicate that the kinase acts by a sequential mechanism. Protection by substrates against thermal inactivation indicates a random order of enzyme interaction with the substrates. The kinase reaction is readily reversible. Upon incubation with ADP, the terminal phosphate group is stoichiometrically recovered as ATP. The reverse reaction is promoted by a variety of other nucleoside diphosphates but not by ATP. Both native and denatured DNA are substrates for the reverse reaction. The reverse and forward reactions can be employed to label 5‘-terminal phosphates without prior dephosphorylation in an exchange reaction. Inorganic sulfate is a relatively strong inhibitor of the kinase; it is competitive with ATP (K1 = 0.2 mM) and noncompetitive with respect to DNA. Dextran sulfate inhibition of the kinase appears competitive with respect to both ATP and DNA. © 1979, American Chemical Society. All rights reserved.