The Enzymatic Phosphorylation of Ribonucleic Acid and Deoxyribonucleic Acid

An enzyme, 5'-hydroxyl polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid and ribonucleic acid in the presence of adenosine triphosphate has been purified from cell-free extracts of Escherichia coli infected with bacteriophage T2. The enzyme activity is also found after infection with bacteriophage T4 but was not detected in extracts of cells infected with T1 or T5. The kinase activity is not formed if cells are infected in the presence of chloramphenicol. The phosphorylation of the 5'-hydroxyl end was established by the following observations. (a) Polynucleotides can be converted to a suitable phosphate acceptor by the action of micrococcal nuclease, an endonuclease which produces 5'-hydroxyl ends in DNA and RNA. (b) The 32P introduced into DNA and RNA by the action of the kinase was quantitatively converted to inorganic phosphate by the action of alkaline phosphatase or phosphomonoesterase. (c) Degradation of 32P-labeled polyriboadenylate and polyribocytidylate, products of the 5'-hydroxyl polynucleotide kinase, with alkali resulted in the quantitative isolation of the 32P as 2'(3')-5'-adenosine diphosphate or 2'(3')-5'-cytidine diphosphate, respectively. No detectable 32P was recovered as 2'(3')-AMP or 2'(3')-CMP. (d) It was shown that tritiated thymidylate introduced at the 3'-hydroxyl end of the DNA chain with DNA polymerase was converted to an acid-soluble form by the action of venom phosphodiesterase much earlier than 32P introduced into DNA by the kinase. Evidence was also presented that the phosphorylation of RNA and DNA by the purified kinase preparation was catalyzed by the same enzyme. The ratio of these two activities is relatively constant during purification and the rates of inactivation of these two activities at 37° were identical.