Molecular Cloning of the Human Gene, PNKP, Encoding a Polynucleotide Kinase 3′-Phosphatase and Evidence for Its Role in Repair of DNA Strand Breaks Caused by Oxidative Damage

Abstract

Polynucleotide kinases catalyze phosphorylation of 5Ј-OH termini of nucleic acids. In a number of biochemical experiments over several decades, evidence for a mammalian polynucleotide kinase (PNK) 1 activities with an acidic pH optimum has mounted (reviewed in Refs. 1-8). We and others have purified such a PNK to near-homogeneity from bovine tissue, which lacks significant 5Ј-phosphorylation activity when assayed with RNA substrates (5, 6, 9). This activity, denoted SNQI-PNK, corresponded to a polypeptide of approximately 60 kDa in our experiments (6). Highly purified SNQI-PNK fractions contain a 3Ј-phosphatase activity (6), originally discovered in the PNK from bacteriophage T4 (10, 11) and also observed in PNKs from rat liver nuclei (2-5, 12). Furthermore, there are reports of mammalian PNK activities with a greater substrate specificity for RNA than DNA (8, 13, 14) 2 and of conservation of yeastlike tRNA ligation (with its requirement for a PNK activity) as a minor pathway in HeLa cells (15).Because of its widespread presence in mammalian cells, the acidic pH optimum PNK is likely to be a key enzyme in DNA metabolism, and its biochemical functions immediately suggest a role in the critical process of DNA repair. One of its enzymatic activities, DNA 3Ј-phosphatase, implies an ability to repair strand breaks terminated by 3Ј-phosphate, a type of DNA damage seen in cells treated with ionizing radiation or hydrogen peroxide (16). Removal of this 3Ј-end blocking lesion allows synthesis by DNA polymerase and joining of nicks by DNA ligase. DNA purified from irradiated thymocytes and irradiated thymus, but not DNA irradiated in vitro, contains strand breaks with 5Ј-OH termini (17, 18). The 5Ј-phosphorylation activity of the SNQI-PNK enzyme suggests a possible model in which 5Ј-OH termini are repaired prior to ligation. 5Ј-OH termini in DNA also occur in ischemia in rat brain (19), after cleavage by nucleases with the appropriate specificity such as DNase II (20), and as intermediates during topoisomerase cleavage (21,22). The highest concentration of 5Ј-DNA termini occurs during DNA replication, and Pohjanpelto and Hölttä (23) proposed that a small fraction of Okazaki fragments contain 5Ј-OH termini; this fraction decreases upon incubation of extracts with ATP at pH 6.0, which was inferred to reflect 5Ј-phosphorylation by a cellular PNK.Despite extensive biochemical studies, to date there are no molecular reagents such as antibodies or cDNAs available for mammalian PNKs, hampering further investigation. We present here the molecular cloning of the PNKP gene, the first gene for a mammalian PNK and the first gene for a DNA-specific kinase from any organism. Concomitantly, the PNKP gene also

A unified view of base excision repair: Lesion-dependent protein complexes regulated by post-translational modification

“…Tyrosyl DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of Top1 from the 3' terminus of DNA, resulting in a gap with a 3'phosphate and a 5'hydroxyl group at the margins [56], although there are alternative mechanisms of repair for this lesion involving Mre11 or Mus81 [57]. After lesion removal by Tdp1, PNKP can then transfer the phosphate group from the 3' to the 5' terminus, thus preparing the DNA ends for repair [58]. If any gapfilling activity is required, pol ß is considered a likely candidate [54,[59][60][61][62].…”

“…Alternatively, the processing of strand-breaks induced by oxidative damage, which includes radiation damage, can be accomplished by PNKP [58]. PNKP converts 3'PO 4 groups to the 3'OH groups necessary for DNA synthesis [58].…”

“…PNKP converts 3'PO 4 groups to the 3'OH groups necessary for DNA synthesis [58]. PNKP has also been implicated in the processing of DNA ends resulting from NEIL glycosylase activity.…”

“…For example, an interaction between XRCC1 and PNKP, a protein necessary for the initial processing of DNA single-strand break termini into ends that are amenable to re-ligation, was shown to be involved in BER following oxidative stress [58,63]. Interestingly, XRCC1 stimulated both the kinase and phosphatase activity of PNKP at damaged termini, resulting in an overall acceleration of single-strand break repair [63], supporting our contention that SSBR may be considered a sub-pathway of BER.…”

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.…”