The Enzymatic Phosphorylation of Ribonucleic Acid and Deoxyribonucleic Acid

Novogrodsky, Abraham; Tal, Moshe; Traub, A.; Hurwitz, Jerard

doi:10.1016/s0021-9258(18)96554-3

Cited by 110 publications

(26 citation statements)

References 17 publications

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“…Various exogenous and endogenous agents including ionizing radiation, chemicals, and nucleases may cause the hydroxylation of the 5′ termini in nucleic acids, resulting in DNA damage and genomic instability. − The 5′-polynucleotide kinase (PNK) is responsible for the repair of the 5′-hydroxyl radical of nucleic acids through transferring the γ-phosphate group from adenosine triphosphate (ATP) to the 5′-hydroxyl group of oligonucleotides or nucleic acids . Malfunction of PNK may cause the deregulation of many cellular activities (e.g., nucleic acid metabolism, DNA replication, DNA recombination, and DNA repair during strand damage), − and eventually induces a variety of human diseases including loom’s syndrome, Werner syndrome, and Rothmund-Thomson syndrome .…”

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confidence: 99%

“…1−4 The 5′-polynucleotide kinase (PNK) is responsible for the repair of the 5′hydroxyl radical of nucleic acids through transferring the γphosphate group from adenosine triphosphate (ATP) to the 5′hydroxyl group of oligonucleotides or nucleic acids. 5 Malfunction of PNK may cause the deregulation of many cellular activities (e.g., nucleic acid metabolism, DNA replication, DNA recombination, and DNA repair during strand damage), 6−9 and eventually induces a variety of human diseases including loom's syndrome, Werner syndrome, and Rothmund-Thomson syndrome. 10 Additionally, PNK is becoming a promising therapeutic target because of the positive effect of 5′-polynucleotide kinase inhibitors in the radio therapy of somatic cancers.…”

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Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles

et al. 2017

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5'-Polynucleotide kinase such as T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5'-hydroxyl termini in nucleic acids, playing a crucial role in DNA replication, DNA recombination, and DNA damage repair. Here, we demonstrate for the first time single-molecule detection of PNK based on phosphorylation-directed recovery of fluorescence quenched by Au nanoparticle (AuNP) in combination with lambda exonuclease-mediated cleavage reaction. In the presence of PNK, the γ-phosphate group from adenosine triphosphate (ATP) is transferred to 5'-hydroxyl terminus, resulting in 5'-phosphorylation of the hairpin probe. The phosphorylated hairpin probes may function as the substrates of lambda exonuclease and enable the removal of 5' mononucleotides from the stem, leading to the unfolding of hairpin structure and the formation of binding probes. The resultant binding probes may specifically hybridize with the AuNP-modified capture probes, forming double-strand DNA (dsDNA) duplexes with 5'-phosphate groups as the substrates of lambda exonuclease and subsequently leading to the cleavage of capture probes and the liberation of Cy5 molecules and the binding probes. The released binding probes may further hybridize with new capture probes, inducing cycles of digestion-release-hybridization and consequently the release of numerous Cy5 molecules. Through simply monitoring Cy5 molecules with total internal reflection fluorescence (TIRF)-based imaging, PNK activity can be quantitatively measured. This assay is very sensitive with a limit of detection of 9.77 × 10 U/μL, and it may be further used to screen the PNK inhibitors and measure PNK in cancer cell extracts.

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Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles

et al. 2017

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“…Concurrently, 1-AR lacks a C-1 hydroxyl, preventing it from being phosphorylated and subsequently converted into NaMN. Ablation of their ability to partake in enzymatic processes potentially increases the probes’ cellular half-lives and reduces background. , …”

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confidence: 99%

An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation

et al. 2020

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Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe’s utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

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“…Phosphorylation of DNA with 5′-hydroxyl termini is an important regulatory process involved in the majority of normal cellular events, including DNA recombination, DNA replication, and DNA repair during strand interruption. − Various exogenous and endogenous agents (e.g., ionizing radiation, chemicals, and nucleases) may induce the hydroxylation of 5′-termini in nucleic acids, resulting in DNA damage and genomic instability. − In this case, polynucleotide kinase (PNK), a kinase that catalyzes the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to the 5′-hydroxyl group of oligonucleotides or nucleic acids, is critical for repairing DNA strand breaks induced by endogenous or exogenous agents. − Malfunctioning PNK is also found to associate with the deregulation of many cellular activities and eventually leads to a variety of human diseases, such as loom’s syndrome, Werner syndrome, and Rothmund–Thomson syndrome . Moreover, PNK is a promising therapeutic target in the radiotherapy of somatic cancers because of the positive effect of PNK inhibitors. , To date, some methods have been reported for PNK assays for its considerable importance in fundamental biochemical and molecular biology studies.…”

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A Dual-Enzyme-Assisted Three-Dimensional DNA Walking Machine Using T4 Polynucleotide Kinase as Activators and Application in Polynucleotide Kinase Assays

et al. 2018

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T4 polynucleotide kinase (T4 PNK), an intracellular kinase, catalyzes the phosphorylation of 5'-hydroxyl termini in nucleic acids and plays a crucial role in DNA-related physiological activities. Malfunctioning of PNK is associated with the deregulation of many cellular activities and eventually induces a variety of human diseases. Herein, we report a smart three-dimensional (3D) DNA walking machine using PNK as an effective activator when coupled with the duplex DNA nuclease-assisted cleavage reaction. The 3D DNA tracks benefit from high DNA loading capacity of gold nanoparticles, and the high efficiency of duplex nuclease-mediated cyclic cleavage facilitates the movement of the DNA machine in response to T4 PNK. The DNA machine is also applied for the PNK assay based on the signal amplification from point to area during the DNA walking process. The method achieves an excellent detection limit of 0.0067 U/mL with a linear range from 0.01 to 0.3 U/mL and a favorable specificity even in complex serum samples. Therefore, the 3D DNA machine shows great potential in biochemical and molecular biology studies, drug discovery, and clinic diagnostics.

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The Enzymatic Phosphorylation of Ribonucleic Acid and Deoxyribonucleic Acid

Cited by 110 publications

References 17 publications

Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles

Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticles

An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation

A Dual-Enzyme-Assisted Three-Dimensional DNA Walking Machine Using T4 Polynucleotide Kinase as Activators and Application in Polynucleotide Kinase Assays

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