Polynucleotide 5'-hydroxyl kinases

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Polynucleotide 5'-hydroxyl kinases ( EC 2.7.1.78 ), often referred to simply as polynucleotide kinases (PNK), are enzymes that transfer the transfer of a phosphate group from the donor ATP to the 5 ' hydroxyl end of a polynucleotide . Like all kinases , they are therefore phospho transferases . They catalyze the reaction:

ATP + 5'-dephospho-DNA ADP + 5'-phospho-DNA

ATP and 5'-dephospho-DNA are the substrates while ADP and 5'-phospho-DNA are the products of the reaction. Polynucleotide kinases thus catalyze the transfer of a gamma phosphate from the ATP to the free hydroxyl end of a DNA or an RNA . Polynucleotide kinases are encoded by bacteriophages such as the T4 phage . Purification and characterization was first achieved in 1965 when examining extracts of the E. coli bacterium that had previously been infected with T4 phages.

Examples

The T4 polynucleotide kinase is an important enzyme in molecular biology , since it allows the labeling of nucleic acids at their 5 'end ( end labeling ) and here in particular of oligonucleotides . Before the establishment of non-radioactive methods, DNA molecules labeled with PNK were, among other things, substrates for sequencing according to Maxam & Gilbert . The ligation of synthetic DNA also requires a 5'-phosphate. Since the reaction is reversible, 5'-phospho-DNA can also be used in the labeling reaction in an exchange reaction. In this case, the reaction is carried out with an excess of γ- 32 P-ATP and ADP. A human polynucleotide kinase was discovered in 1999. The protein, polynucleotide kinase 3'- phosphatase , a bifunctional protein which has a 3'-phosphatase activity in addition to 5'-kinase activity, plays a role in DNA repair .

Individual evidence

  1. ↑ CC Richardson: Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. In: Proceedings of the National Academy of Sciences . Volume 54, Number 1, July 1965, pp. 158-165, PMID 5323016 , PMC 285814 (free full text).
  2. ↑ A. Novogrodsky, J. Hurwitz: The enzymatic phosphorylation of ribonucleic acid and deoxyribonucleic acid. I. Phosphorylation at 5'-hydroxyl termini. In: The Journal of biological chemistry. Volume 241, Number 12, June 1966, pp. 2923-2932, PMID 4287929 .
  3. ↑ A. Novogrodsky, M. Tal, A. Traub, J. Hurwitz: The enzymatic phosphorylation of ribonucleic acid and deoxyribonucleic acid. II. Further properties of the 5'-hydroxyl polynucleotide kinase. In: The Journal of biological chemistry. Volume 241, Number 12, June 1966, pp. 2933-2943, PMID 4287930 .
  4. ↑ AM Maxam, W. Gilbert: Sequencing end-labeled DNA with base-specific chemical cleavages. In: Methods in enzymology. Volume 65, Number 1, 1980, pp. 499-560, PMID 6246368 .
  5. ↑ A. Jilani, D. Ramotar, C. Slack, C. Ong, XM Yang, SW Scherer, DD Lasko: Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage. In: The Journal of biological chemistry. Volume 274, Number 34, August 1999, pp. 24176-24186, PMID 10446192 .