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

The recombinant human bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is unique in its absolute requirement for Mg2+ and inorganic phosphate. Both ions affect the affinity of the enzyme for NAD and have no effect on the binding of methylenetetrahydrofolate. The NAD cofactor can be replaced by NADP with a much higher KM and lower VMAX. Kinetic investigation using NADP supports the role of Mg2+ in dinucleotide binding and illustrates that the 2'-phosphate can substitute for phosphate in this process. The human NAD-dependent bifunctional enzyme has a 44% amino acid sequence identity with the dehydrogenase-cyclohydrolase domain of the yeast mitochondrial NADP-dependent trifunctional enzyme encoded by the MIS1 gene, compared to 37% identity with the corresponding domain of the cytosolic trifunctional enzyme. The sequence comparison and the kinetic properties suggest that the NAD bifunctional enzyme is the mammalian homolog of the yeast mitochondrial trifunctional enzyme, which has evolved a unique use of inorganic phosphate to change its dinucleotide specificity from NADP to NAD. Its role is proposed to be in providing formyltetrahydrofolate for the synthesis of formylmethionyl transfer RNA required for the initiation of protein synthesis in mitochondria.

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Europe PMC (https://europepmc.org) is a database of research articles, including peer reviewed full text articles and abstracts, and preprints - all freely available for use via website, APIs and bulk download. This article outlines new developments since 2017 where work has focussed on three key areas: (i) Europe PMC has added to its core content to include life science preprint abstracts and a special collection of full text of COVID-19-related preprints. Europe PMC is unique as an aggregator of biomedical preprints alongside peer-reviewed articles, with over 180 000 preprints available to search. (ii) Europe PMC has significantly expanded its links to content related to the publications, such as links to Unpaywall, providing wider access to full text, preprint peer-review platforms, all major curated data resources in the life sciences, and experimental protocols. The redesigned Europe PMC website features the PubMed abstract and corresponding PMC full text merged into one article page; there is more evident and user-friendly navigation within articles and to related content, plus a figure browse feature. (iii) The expanded annotations platform offers ∼1.3 billion text mined biological terms and concepts sourced from 10 providers and over 40 global data resources.