Polynucleotide Kinase Assays

Weinfeld M, Liuzzi M, Paterson MC (1990) Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29 1737-1743... 

Restriction-site mapping in DNA may be performed by end-labelling with polynucleotide kinase as above, and subsequent partial digestion with the restriction enzyme. An overlapping series of polynucleotides with a common labelled terminus is thus formed, and size analysis of these affords a restriction map. Polynucleotide kinase may also be used to assay the number of 5 -phosphorylated termini in DNA generated as the result of the action of restriction endonuclease. In the presence of... 

Restriction enzyme activities can be assayed by various methods including gel visualization assay, polynucleotide kinase exchange assay (36), and exonuclease-coupled assay (37). By far the most common of all assay methods is the visualization by EtBr staining of the digestion products separated by electrophoresis in an agarose gel (30,38). This method is straightforward and enables the detection of contaminating activities of endo- and exonucleases. 

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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