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

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has diverse biological functions including its nuclear translocation in response to oxidative stress. We show that GAPDH physically associates with APE1, an essential enzyme involved in the repair of abasic sites in damaged DNA, as well as in the redox regulation of several transcription factors. This interaction allows GAPDH to convert the oxidized species of APE1 to the reduced form, thereby reactivating its endonuclease activity to cleave abasic sites. The GAPDH variants C152G and C156G retain the ability to interact with but are unable to reactivate APE1, implicating these cysteines in catalyzing the reduction of APE1. Interestingly, GAPDH-small interfering RNA knockdown sensitized the cells to methyl methane sulfonate and bleomycin, which generate lesions that are repaired by APE1, but showed normal sensitivity to 254-nm UV. Moreover, the GAPDH knockdown cells exhibited an increased level of spontaneous abasic sites in the genomic DNA as a result of diminished APE1 endonuclease activity. Thus, the nuclear translocation of GAPDH during oxidative stress constitutes a protective mechanism to safeguard the genome by preventing structural inactivation of APE1.The evolutionary conserved enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 3 exists as a tetramer that catalyzes a critical reaction in the second stage of the glycolytic pathway (1). It uses the oxidized form of nicotinamide adenine dinucleotide (NAD ϩ ) and converts glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with the concomitant release of NADH in an oxido-reduction reaction (1). GAPDH is also a key redox-sensitive protein that possesses an active site cysteine sulfhydryl that is susceptible to oxidation (2). Under oxidative stress, GAPDH rapidly undergoes disulfide bond formation leading to reduction in its enzymatic activity (2, 3). GAPDH has the propensity to interact with several proteins that are vulnerable to aggregation and are associated with neurodegenerative disorders such as in the case of the pro-oxidant amyloid ␤ peptide involved in Alzheimer disease (4). Recent studies have documented that GAPDH is also involved in several other nuclear processes that include histone H2B gene expression, nuclear RNA export, apoptosis, and cellular response to DNA damage (5-8).Several lines of evidence support a role for GAPDH in DNA damage and repair (5, 9). For example, GAPDH can translocate from the cytoplasm to the nucleus when cells are challenged with the potent chemical oxidant and DNA-damaging agent H 2 O 2 , although it is not clear what is the function executed by GAPDH under this stress condition (10). However, a more recent study documented that nitric oxide can also induce nuclear localization of GAPDH where it is acetylated by the acetyltransferase p300/CBP via direct protein interaction, which in turn causes stimulation of the catalytic activity of p300/CBP, resulting in the activation of downstream targets such as p53 (11). Other studies have shown that GAPDH is associated w...

Tpp1 is a DNA 3-phosphatase in Saccharomyces cerevisiae that is believed to act during strand break repair. It is homologous to one domain of mammalian polynucleotide kinase/3-phosphatase. Unlike in yeast, we found that Tpp1 could confer resistance to methylmethane sulfonate when expressed in bacteria that lack abasic endonuclease/3-phosphodiesterase function. This species difference was due to the absence of ␦-lyase activity in S. cerevisiae, since expression of bacterial Fpg conferred Tpp1-dependent resistance to methylmethane sulfonate in yeast lacking the abasic endonucleases Apn1 and Apn2. In contrast, ␤-only lyases increased methylmethane sulfonate sensitivity independently of Tpp1, which was explained by the inability of Tpp1 to cleave 3 ␣,␤-unsaturated aldehydes. In parallel experiments, mutations of TPP1 and RAD1, encoding part of the Rad1/Rad10 3-flap endonuclease, caused synthetic growth defects in yeast strains lacking Apn1. In contrast, Fpg expression led to a partial rescue of apn1 apn2 rad1 synthetic lethality by converting lesions into Tpp1-cleavable 3-phosphates. The collected experiments reveal a profound toxicity of strand breaks with irreparable 3 blocking lesions, and extend the function of the Rad1/Rad10 salvage pathway to 3-phosphates. They further demonstrate a role for Tpp1 in repairing endogenously created 3-phosphates. The source of these phosphates remains enigmatic, however, because apn1 tpp1 rad1 slow growth could be correlated with neither the presence of a yeast ␦-lyase, the activity of the 3-phosphate-generating enzyme Tdp1, nor levels of endogenous oxidation.