T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the γ-phosphate from ATP to the 5′-terminus of polynucleotides or to mononucleotides bearing a 3′-phosphate group. In addition to this 5′-kinase activity, T4 PNK also has 3′-phosphatase activity, which converts 3′-phosphate ends to 3′-hydroxyl termini. Under certain conditions, the reaction is reversible, permitting exchange of the γ-phosphate of ATP with the 5′-terminal phosphate of a polynucleotide.