Polynucleotide Kinase as a Potential Target for Enhancing Cytotoxicity by Ionizing Radiation and Topoisomerase I Inhibitors

Bernstein, Nina; Karimi‐Busheri, Feridoun; Rasouli-Nia, Aghdass; Mani, Rajam S.; Dianov, Grigory L.; Glover, J. N. Mark; Weinfeld, Michael

doi:10.2174/187152008784220311

Cited by 31 publications

(24 citation statements)

References 86 publications

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“…Phage T4 polynucleotide kinase and hPNKP share similar nucleic acid kinase and phosphatase activities. However, with the exception of the enzyme active sites, the proteins bear no recognizable homology (26,28). The phosphatase domains of both proteins belong to the haloacid dehalogenase superfamily (26,39,40) with a conserved DxDGT motif, where the first Asp forms a covalent phospho-aspartate intermediate with the substrate.…”

Section: Discussionmentioning

confidence: 99%

“…It has recently been suggested that PNKP could be a potential target for small molecule inhibitors (2,28). We report here the development of a fluorescence-based assay for screening chemical libraries for inhibitors of the phosphatase activity of PNKP and its application in the screening of a library of drug-like polysubstituted piperidines.…”

Section: Introductionmentioning

confidence: 99%

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Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

et al. 2009

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Human polynucleotide kinase/phosphatase (hPNKP) is a 57.1-kDa enzyme that phosphorylates DNA 5 ¶-termini and dephosphorylates DNA 3 ¶-termini. hPNKP is involved in both single-and double-strand break repair, and cells depleted of hPNKP show a marked sensitivity to ionizing radiation. Therefore, small molecule inhibitors of hPNKP should potentially increase the sensitivity of human tumors to ;-radiation. To identify small molecule inhibitors of hPNKP, we modified a novel fluorescence-based assay to measure the phosphatase activity of the protein, and screened a diverse library of over 200 polysubstituted piperidines. We identified five compounds that significantly inhibited hPNKP phosphatase activity. Further analysis revealed that one of these compounds, 2-(1-hydroxyundecyl)-1-(4-nitrophenylamino)-6-phenyl-6,7a-dihydro-1H-pyrrolo[3,4-b]pyridine-5,7(2H,4aH)-dione (A12B4C3), was the most effective, with an IC 50 of 0.06 Mmol/L. When tested for its specificity, A12B4C3 displayed no inhibition of two well-known eukaryotic protein phosphatases, calcineurin and protein phosphatase-1, or APTX, another human DNA 3 ¶-phosphatase, and only limited inhibition of the related PNKP from Schizosaccharomyces pombe. At a nontoxic dose (1 Mmol/L), A12B4C3 enhanced the radiosensitivity of human A549 lung carcinoma and MDA-MB-231 breast adenocarcinoma cells by a factor of two, which was almost identical to the increased sensitivity resulting from shRNA-mediated depletion of hPNKP. Importantly, A12B4C3 failed to increase the radiosensitivity of the hPNKP-depleted cells, implicating hPNKP as the principal cellular target of A12B4C3 responsible for increasing the response to radiation. A12B4C3 is thus a useful reagent for probing hPNKP cellular function and will serve as the lead compound for further development of PNKP-targeting drugs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

et al. 2009

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“…Substrate binding was stabilized by mutation of Asp170 to alanine (PNKP D170A ). Asp170 is predicted to attack the 3′-phosphate, generating a covalent phosphoaspartate intermediate in the first step of the reaction (13). We were successful in crystallizing this domain in the apo form, and bound to five nucleotide single-stranded DNAs bearing either C, T, or A at the 3′ position, and these structures were determined to resolutions between 1.7 and 2.1 Å (Table S1, Materials and Methods, and Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The PNKP phosphatase domain adopts a haloacid dehydrogenase (HAD) fold (14) commonly found in many phosphatases, and contains conserved active site residues that suggest the enzyme utilizes a two step mechanism that is characteristic of this family (13). Key to catalysis is an active site Mg þþ ion, which binds and stabilizes the growing negative charge on the substrate phosphate during catalysis, as well as binding and positioning one of the critical active site aspartate residues.…”

Section: Resultsmentioning

confidence: 99%

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates

Coquelle

Havali-Shahriari

Bernstein

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5′-phosphate and 3′-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3′-phosphate termini within DNA nicks, gaps, or at double-or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3′-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.DNA substrate recognition | HAD family | | protein-DNA complexes | X-ray crystallography D NA damage induced by ionizing radiation, as well as DNA repair intermediates generated during base excision repair, often result in the formation of DNA ends containing 3′-phosphate/5′-hydroxyl termini. Because DNA polymerases and ligases require 3′-hydroxyl/5′-phosphate termini to complete DNA repair, cells have evolved enzymes to recognize and process these damaged DNA ends. Mammalian polynucleotide kinase/phosphatase (PNKP) contains 3′-phosphatase and 5′-kinase activities present in two distinct active sites (1). The critical role of PNKP in various DNA repair processes is underlined by the fact that RNAi knockdown of PNKP leads to marked sensitization of human cells to spontaneous mutation as well as to various genotoxic agents (2). A particularly important role is attributed to the phosphatase activity as small molecules that selectively inhibit the phosphatase but not the kinase activity of PNKP sensitize human cells to DNA damage in a PNKP-dependent manner (3, 4). Mutations in the PNKP phosphatase domain are associated with the developmental neurological disorder MCSZ, revealing a critical role for PNKP in human neurological development (5).The PNKP phosphatase domain plays a central role in several critical DNA repair processes. For example, PNKP participates in base excision repair (BER) in partnership with the NEIL (endonuclease VIII-like) DNA glycosylases. NEILs exhibit β,δ-AP (aminopurine) lyase activity, which produces single nucleotide gap DNAs containing 3′-phosphate termini that are subsequently dephosphorylated by PNKP prior to gap filling and ligation (6, 7). PNKP is also associated with the repair of double-strand breaks through the nonhomologous end joining (NHEJ) pathway (8, 9), in a manner that relies on the interaction of PNKP with the NHEJ scaffolding protein, XRCC4 (10). Consistent with its role in these DNA repair pathways, PNKP recognizes 3′-phosphate termini ...

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“…This stage of processing in the repair by NHEJ is believed to be responsible for errors resulting from losses and additions of nucleotides at the sites of DNA DSB rejoining (Mahaney et al, 2009;Lieber, 2010). In the processing of broken DNA ends, the following enzymes are involved: the nuclease Artemis (Yannone et al, 2008), the DNA polymerases of the family X (Pol and Pol ) (Moon et al, 2007), and the terminal deoxynucleotidyl transferase (Mahaney et al, 2009), as well as polynucleotide kinase having both the kinase and phosphatase activities (Bernstein et al, 2008). Also, some other substances participate in the process, such as aprataxin (APTX) which catalyzes the removal of AMP groups at the 5 'ends of the DSB (Rass et al, 2008), the PALF-factor, and the protein cernunnos (XLF), which interact with XRCC4 in the activation of the DNA-ligase complex XRCC4/DNK-ligase IV/XLF Yano et al, 2009).…”

Section: Repair Of Dna Double Strand Breaksmentioning

confidence: 99%

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

Gaziev¹,

Shaikhaev²

2012

Current Topics in Ionizing Radiation Research

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Polynucleotide Kinase as a Potential Target for Enhancing Cytotoxicity by Ionizing Radiation and Topoisomerase I Inhibitors

Cited by 31 publications

References 86 publications

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Identification of a Small Molecule Inhibitor of the Human DNA Repair Enzyme Polynucleotide Kinase/Phosphatase

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates

Limited Repair of Critical DNA Damage in Cells Exposed to Low Dose Radiation

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