T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini

Abstract

The amount of reaction catalyzed by T4 polynucleotide kinase on a variety of its substrates is greatly increased in the presence of polyethylene glycol 8000 (PEG 8000). Both the forward and reverse reactions as well as the exchange reaction can be stimulated. The stimulation is a general effect on T4 polynucleotide kinase reactions involving high molecular weight DNA substrates. The use of PEG 8000 is particularly advantageous for labeling or removing terminal 5'-phosphate groups which are only slowly or incompletely labeled or removed under ordinary conditions, such as those at recessed termini or at "nicks" in duplex DNA, although the reaction on blunt-ended or protruding termini is also increased. It is further advantageous for labeling very low concentrations of substrates.