Polynucleotide Kinase

Kleppe, Kjell

doi:10.1002/9780470122938.ch5

Cited by 8 publications

(2 citation statements)

References 54 publications

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“…The 32 P-labeling (to γ-phosphate group of ATP) is a well-established direct method for PNK assay. − With the labels transferring from ATP donor to DNA acceptor, the increase of 32 P-labeled DNA or the decrease of ATP 32 can be easily observed . However, this method is limited by its laborious, discontinuous monitoring and the requirement of radioactive labels.…”

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confidence: 99%

A Label-Free Bioluminescent Sensor for Real-Time Monitoring Polynucleotide Kinase Activity

et al. 2014

Anal. Chem.

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Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5'-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.

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confidence: 99%

A Label-Free Bioluminescent Sensor for Real-Time Monitoring Polynucleotide Kinase Activity

et al. 2014

Anal. Chem.

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“…At present, the function of mammalian DNA kinase and T4 polynucleotide kinase iiz vivo remains unclear (see [17]). It seems likely that DNA kinase plays an important role in repair of damaged DNA and in replication in co-operation with DNA ligase.…”

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confidence: 99%

Characterization of DNA Kinase from Calf Thymus

Tamura

Teraoka

Tsukada

2005

European Journal of Biochemistry

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of at DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity 16.7 units/mg protein at 25 "C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 x lo4. The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 x lo' and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate ( K , = 7 m M ) and pyrophosphate ( K , = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.

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