INFLUENCES OF MACROMOLECULAR CROWDING ON BIOCHEMICAL SYSTEMS

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T4 polynucleotide kinase rapidly loses activity during its reaction on duplex DNA termini. Addition of high concentrations of nonspecific polymers reverses or prevents this inactivation. In contrast, additions of related materials of lower molecular weight are relatively ineffective in stabilizing the kinase. Such a pattern suggests that the stabilizing effects of polymers on kinase activity are due to macromolecular crowding. An effect of crowding on the known tendency of the kinase to undergo oligomerization reactions is consistent with our observations. This effect of polymers on the kinase can be exploited to greatly increase the amount of reaction obtainable on 5'-hydroxyl groups of duplex DNA substrates located at recessed or blunt ends or at "nicks" which are otherwise difficult to effectively phosphorylate or dephosphorylate. Macromolecular crowding was found to cause no striking change in the processivity of the T4 DNA ligase under several reaction conditions. Interactions with the Patent Office in connection with an application on behalf of the Government have been successfully concluded and we await the issuance of a patent based on our previous studies of polymer-stimulated ligation of DNA.