Quantitation of the various termini generated by type II restriction endonucleases using the polynucleotide kinase exchange reaction.

ABSTRACT

Parameters of the polynucleotide kinase-catalyzed exchange reaction between [gamma-32P]ATP and 5'-phosphoryl DNAs have been measured with the termini generated by the following endonucleases EcoRI (Berkner, K. L., and Folk, W. R. (1977) J. Biol. Chem. 252, 3176-3184), Hpa II, BamHI, and HindIII (external termini); HindII and Hpa I (blunt terminal); Hae II and Hha I (internal termini). In every case, exchange is reproducible and proportional to the number of termini. However, in most cases, the exchange reaction does not proceed to the theoretical maximum. External termini and single-stranded DNAs are labeled more rapidly and to approximately 5-fold higher levels than blunt or internal termini. Concentrations of 100 to 200 microns ADP and 12 microns ATP are optimal for labeling all types of termini with the exchange reaction.