Purification of a polynucleotide kinase from calf thymus, comparison of its 3′-phosphatase domain with T4 polynucleotide kinase, and investigation of its effect on DNA replication in vitro

Mammalian polynucleotide kinases (PNKs) carry out 5′‐phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3′‐phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3′‐phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI‐PNK, with a slightly acidic pH optimum that copurifies with a 3′‐phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI‐PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3′‐phosphatase activity of T4 PNK to the C‐terminus and suggest that the 3′‐phosphatase domain of T4 PNK is related to the protein superfamily of l‐2‐haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI‐PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme. J. Cell. Biochem. 73:188–203, 1999. © 1999 Wiley‐Liss, Inc.