Polynucleotide kinase exchange reaction

Procedure D is a method for 5 -end labelling of a set of fragments, not previously dephosphorylated, using the polynucleotide kinase catalysed exchange reaction. 

An alternative to the two-stage end-labeling protocol involves a phosphate exchange reaction catalyzed by polynucleotide kinase. In the presence of excess amounts of ADP, polynucleotide kinase transfers a 5 -phosphoryl residue from DNA or RNA to ADP in a reversal of the phosphorylation reaction (5,12). At pH... 

Photochemical cross-linking studies of ribosomal RNA and a tRNA derivative have been achieved using the 5 -[Y- P]-labelled 3 , 5 -bisphosphate of 2, 6-diazido-9-(P-D-ribofuranosyl)purine. The compound was prepared by reaction of the corresponding nucleoside with pyrophosphoryl chloride followed by exchange of the 5 -phosphate with the y-phosphate of [ y- 2p]j jp catalysed by T4 polynucleotide kinase. It was then enzymatically ligated to the 3 -terminus of a yeast tRNA he molecule. 

Fig. 10. 5 End labeling of DNA and RNA. Labeling is most efficient with single- stranded molecules and at the 5 ends of double-stranded DNA with recessed 3 ends. Blunt-ended DNA and DNA with recessed 5 ends react more slowly. After removal of the S phosphate with alkaline phosphatase, the S OH group is rephosphorylated by the action of polynucleotide kinase and ATP. Alternatively, in the presence of excess ADP, the reverse reaction is encouraged, effectively leading to exchange between then 5 phosphate and the y-phosphate of ATP.

Polynucleotide phosphorylase can be assayed in several ways. The liberation of inorganic phosphate can be used to follow the reaction. Both disappearance of acid-soluble nucleotides and formation of acid-insoluble nucleotides have been measured. The reverse reaction in the presence of P Mabeled phosphate allows an exchange reaction, the incorporation of P into nucleotides, to be used as an assay. The exchange reaction was the first reaction of this enzyme to be discovered, and studies on the responsible enzyme led to the finding of polynucleotide synthesis. Another assay based on the reverse reaction has been devised for rapid spectrophoto-metric determinations. A polymer of adenylic acid is incubated with the phosphorylase in the presence of phosphate, phosphopyruvate, pyruvate kinase, DPNH, and lactic dehydrogenase. The formation of ADP is thus coupled with the formation of pyruvate which reacts stoichiometrically with PPNH, so that the entire reaction can be followed at 340 m/a. 

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