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
✍ Scribed by Arshad Jilani; Carolyn Slack; Diamanto Matheos; Maria Zannis-Hadjopoulos; Dana D. Lasko
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 148 KB
- Volume
- 73
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
Mammalian polynucleotide kinases (PNKs) carry out 5Ј-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 3Ј-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3Ј-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3Ј-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 3Ј-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3Ј-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.