Molecular Characterization of a Human DNA Kinase

Abstract

Human polydeoxyribonucleotide kinase is an enzyme that has the capacity to phosphorylate DNA at 5-hydroxyl termini and dephosphorylate 3-phosphate termini and, therefore, can be considered a putative DNA repair enzyme. The enzyme was purified from HeLa cells. Amino acid sequence was obtained for several tryptic fragments by mass spectrometry. The sequences were matched through the dbEST data base with an incomplete human cDNA clone, which was used as a probe to retrieve the 5-end of the cDNA sequence from a separate cDNA library. The complete cDNA, which codes for a 521-amino acid protein (57.1 kDa), was expressed in Escherichia coli, and the recombinant protein was shown to possess the kinase and phosphatase activities. Comparison with other sequenced proteins identified a P-loop motif, indicative of an ATP-binding domain, and a second motif associated with several different phosphatases. There is reasonable sequence similarity to putative open reading frames in the genomes of Caenorhabditis elegans and Schizosaccharomyces pombe, but similarity to bacteriophage T4 polynucleotide kinase is limited to the kinase and phosphatase domains noted above. Northern hybridization revealed a major transcript of approximately 2.3 kilobases and a minor transcript of approximately 7 kilobases. Pancreas, heart, and kidney appear to have higher levels of mRNA than brain, lung, or liver. Confocal microscopy of human A549 cells indicated that the kinase resides predominantly in the nucleus. The gene encoding the enzyme was mapped to chromosome band 19q13.4.Transient DNA strand breaks and short gaps are frequently observed in cellular DNA. Many arise during regular cellular activity such as DNA replication, recombination, or differentiation. Others occur as a consequence of exposure to endogenous or exogenous DNA damaging agents. Repair of these strand interruptions is usually mediated by DNA ligases and polymerases. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by nucleases, such as DNase II, and many produced by ionizing radiation bear 3Ј-phosphate and 5Ј-hydroxyl groups (1-4), and therefore must be processed before they can be acted upon by DNA ligases or polymerases.One enzyme that possesses the capacity to both phosphorylate 5Ј-hydroxyl termini and dephosphorylate 3Ј-phosphate termini is polynucleotide kinase (PNK).1 The PNK from T4 phage has found widespread application in molecular biology, especially for radiolabeling DNA and oligonucleotides (5). It can act on DNA and RNA and even phosphorylate nucleoside 3Ј-monophosphates. However, the main cellular function of the T4 enzyme is not to repair DNA, but rather to counter the action of a phage endoribonuclease that cleaves tRNA (6). Eukaryotic PNKs fall into two categories depending on whether their preferred substrate is DNA or RNA (7). While both can phosphorylate 5Ј-termini, only the former have an associated 3Ј-phosphatase activity (8 -12).Mammalian DNA kinases have ...

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

“…Residues 6 through 102 of the N-terminus of Aprataxin are 41% identical with the N-terminus of human polynucleotide kinase (PNK), an enzyme with 5′-DNA kinase, 3′-phosphatase activity that is located C-terminal to this domain (33,34 Many eukaryotes appear to have multiple Hint family members, one or more of which are Hint homologs, and one which is an Aprataxin homolog (Table 1). These sequences can be distinguished by degree of similarity to Hint and Aprataxin.…”

Regulation and mechanisms of mammalian double-strand break repair

“…PNKP can remove 3 0 -phosphates from blunt, recessed or protruding 3 0 termini (Jilani et al, 1999;Karimi-Busheri et al, 1999). PNKP also phosphorylates 5 0 -hydroxyl ends, and this phosphorylation appears to be coupled to the end-joining process (Chappell et al, 2002).…”

Stable down-regulation of human polynucleotide kinase enhances spontaneous mutation frequency and sensitizes cells to genotoxic agents

“…The cDNA for hPNK encodes a 57.1-kDa monomeric protein (3)(4)(5). A role for mammalian PNK in DNA repair has been espoused ever since its discovery (6)(7)(8), and more recently, the need for such an enzyme has been reiterated by those examining the actions of topoisomerase I inhibitors (9, 10).…”

“…Human polynucleotide kinase (hPNK) is a bifunctional enzyme with DNA 5Ј-kinase and 3Ј-phosphatase activities (3,4). The cDNA for hPNK encodes a 57.1-kDa monomeric protein (3)(4)(5).…”