T4 Polynucleotide Kinase: Definition & Overview

DNA strand with two bandaids

Definition of T4 Polynucleotide Kinase

Nucleic acids can be modified at the polynucleotide level through many enzymatic reactions. It includes methylation of both DNA and RNA, glycosylation of DNA, and a nucleotide addition to DNA and RNA molecules at 3′-hydroxyl termini. 

Polynucleotide kinases (PNK) are one such DNA modification enzymes that add phosphate groups to nucleic acid molecules. They transfer the gamma phosphate group from adenosine triphosphate (ATP) to the 5’ hydroxyl termini of DNA or RNA. Additionally, they also perform phosphatase activities in some situations.

The enzyme was first discovered by Richardson and Hurwitz laboratories in Escherichia coli (or E. coli) that was infected with T4 and T2 bacteriophages. It belongs to the family of transferases, specifically those that transfer phosphorus-containing groups to alcohol groups (phosphotransferases). Systematically, the enzyme class is known as ATP:5′-diphospho polynucleotide 5′-phosphotransferase.

T4 PNK has a molecular weight of 132 kDa. It’s a tetramer of identical monomers with each having a molecular weight of 33 kDa and no kinetic cooperativity. 

The dual roles—kinase and phosphatase—performed by the enzyme reside in its separate domains. The N-terminal is a 5′-kinase domain with a nucleotide-binding motif GXXXXGK(S/T) and a C-terminal is a 3′-phosphohydrolase domain.

PNK is an essential tool in labs to study gene expression, DNA modification, or cloning of organisms. Though the Polynucleotide Kinase family includes PNKs from a variety of organisms, typically, the enzyme used in the lab is purified from Escherichia coli B (suppressor-minus) cells infected with phage T4.

The inhibitors of T4 Polynucleotide Kinase (PNK) are ammonium ions, KCL or NaCl, and low levels of phosphate buffers. Moreover, the addition of EDTA or heating the enzyme at 70°C for 15 minutes causes the inactivation of T4 PNK activity.

In this article, we will cover the functions and mechanism of T4 Polynucleotide Kinases, their applications, and quality control tests that are performed to check the efficacy of manufactured T4 Polynucleotide Kinase products.

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Functions of T4 Polynucleotide Kinase

The Polynucleotide Kinase (PNK) has dual activities: kinase and phosphatase. These functions depend on the reaction condition of the organisms. 

Below is a list of roles performed by PNK in different organisms:

  • The enzyme is involved in phosphorylating nucleic acid termini. The PNK reaction in this situation is represented as:

ATP + 5′-dephospho-DNA ⇌ ADP + 5′-phospho-DNA

  • T4 PNK is involved in repairing broken RNA and DNA termini. It does so by converting 3′ PO4/5′ OH ends into 3′ OH/5′ PO4 ends and making them suitable to be joined by DNA and RNA ligases. 

For example, in metazoans and fission yeast, damaged DNA due to oxidation, radiation, and topoisomerase I poison is repaired by the specific activity of 5′ OH phosphorylation of PNK orthologs. 

  • PNK phosphatase is essential for DNA end processing during its damage repair due to oxidative stress or topoisomerase I poison. The enzyme can do both, processing of 3’-phosphate and 5’-hydroxyl termini.
  • During T4 infection bacteria try to protect themselves by inducing site-specific breakage of host-cell tRNAs. However, T4 PNK in phage repair the broken tRNAs and facilitate T4 infection. The enzymatic reaction involves two steps: 
  • Transfer of the γ phosphate from ATP to the RNA’s 5′ OH terminus.
  • Hydrolysis of the 3′ PO4 terminus of RNA.
  • Any mutation in PNK phosphatase, in a region that’s adjacent to the kinase domain, leads to early-onset intractable seizures, autosomal-recessive microcephaly, and developmental delay

What Fields Use T4 PNK?

T4 PNK causes the phosphorylation at the termini of nucleic acids and the resulting end product from this reaction is used for many experimental purposes.

The two most significant areas having the application of T4 PNK include:

  • Molecular biology: T4 PNK is widely used to phosphorylate 5’end of both DNA and RNA in the labeling and ligation experiments. In addition, it’s also used for cloning purposes.
  • Biochemistry: Apart from adding phosphate groups to nucleic acids, it’s also used to remove 3′phosphoryl groups from nucleotide/nucleoside or DNA and RNA to study gene expressions and gene functions. Moreover, it’s used to understand different nucleic acid reactions in a variety of organisms.

T4 PNK Applications

The application of T4 PNK in labeling the 5′ end of nucleic acids with 32P paved the way for advances in molecular cloning, structure analysis, and nucleic acid sequencing.

The enzyme is used in labs along with other reagents based on the experimentations, including DNA polymerase, T4 DNA ligase, 1X T4 Polynucleotide Kinase Reaction Buffer, and storage buffer which is prepared by adding:

  • 10 mM Tris-HCl
  • 50 mM KCl
  • 1 mM DTT
  • 0.1 mM EDTA
  • 50% Glycerol
  • 0.1 µM ATP
  • pH 7.4 @ 25°C

The enzyme is commercially available in units and its understanding helps to determine the amount of enzymes required to be used for your experiment. 

Unit definition:  One unit of T4 Polynucleotide Kinase enzyme is defined as the amount of T4 PNK required to catalyze the transfer of 1 nmol of phosphate from  [γ-32P]ATP to the 5´-OH end of a polynucleotide in 30 minutes at 37°C.

Below is a list of lab assays that involved the use of T4 PNK

  • For labeling 5’ termini of DNA and RNA, which is used as:
  1. Hybridization probe
  2. Transcript mapping probe
  3. Gel electrophoresis marker
  4. DNA sequencing Primer
  5. PCR primer
  • Oligonucleotide synthesis
  • RNA amplification and labeling
  • 5′-phosphorylation of oligonucleotides, PCR products, and other DNA or RNA before ligation
  • Phosphorylation of PCR primers
  • Detection of DNA modification by the [32P]-post labeling assay
  • Removal of 3′-phosphate groups
  • 5′ end-labeling of 3′ phosphorylated oligos

Labeling of Nucleic Acids

T4 PNK is most commonly used in labs for labeling purposes. It’s widely used for the end-labeling of DNA, RNA, short oligonucleotide probes, and other molecules. 

A reversible PNK reaction is also possible, as this would enable nucleotides to be dephosphorylated without using alkaline phosphatases.

Phosphorylation

T4 PNK causes phosphorylation of a variety of molecules including oligonucleotides, single- and double-stranded DNAs and RNAs, and nucleoside 3′-monophosphates before ligation and cloning experiments.

Using polyethylene glycol (PEG) and spermidine in the exchange reaction mixture improves and makes the phosphorylation reaction more efficient.

Polynucleotide Kinase is also used to catalyze the dephosphorylation of deoxynucleoside 3´-monophosphates, 3´-phosphoryl polynucleotides, and deoxynucleoside 3´-diphosphates.

QC Tests

The manufactured T4 Polynucleotide Kinase must not contain RNase, exo-, or endonuclease molecules. The manufactured product should be >95% pure, which is checked by SDS polyacrylamide gel electrophoresis followed by BlueSafe staining.

Additionally, the reactions performed for quality control of the manufactured enzyme are given below.

Endonuclease

The nuclease assay is performed to test the DNase and RNase contamination. 

  • For the DNase contamination test, 10 Units of T4 Polynucleotide Kinase are incubated with  0.2-0.3 μg of supercoiled pNZY28 recombinant plasmid DNA for 14-16 hours at 37 °C.
  • For the RNase contamination test, 10 Units of T4 Polynucleotide Kinase are incubated with 1 µg of RNA for 1 hour at 37 °C. 

After incubation, the nucleic acids are visualized on a GreenSafe-stained agarose gel to check for breaks in the nucleic acids. You must not observe any nicks in the nucleic acid sample if the enzyme is of pure and of high quality.

End-labeling

In this method, DNA is dephosphorylated before performing the labeling reaction. It is more effective to label protruding 5’ ends rather than blunt or recessed 5’ ends. 

Cloning Assay

In this experiment, the T4 Polynucleotide Kinase activity is determined by phosphorylating DNA to be inserted in a non-phosphorylated vector. An evaluation of Kinase’s reaction is made by counting how many bacterial colonies are transformed.

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Polynucleotide Kinase is an enzyme involved in phosphorylation and dephosphorylation of nucleic acids. It also has an essential role in repairing DNA nicks and gaps by making nucleic acids an appropriate substrate for the repair reaction. 

The end product resulting from the Polynucleotide Kinase reaction is utilized in molecular biology and biochemistry labs for multiple purposes, including oligos synthesis, cloning, and nucleic acid end labeling.

For these experiments, a high-quality PNK is required and the quality of the commercially available PNK enzymes is tested through endonuclease, end-labeling, and cloning assays. However, along with high-quality reagents, high-throughput instruments are also required to obtain validated results and eliminate duplicate experiments.

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