Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol

Abstract

Monovalent cations such as Na+ and K+ inhibit the activity of T4 DNA ligase. However, the extent of inhibition varies with the terminal sequence of the duplex DNA used as substrate; in many cases, ligation of DNA is completely inhibited at 200 mM. The activity of the ligase is stimulated by raising the concentration of polyethylene glycol 6000 from 0 to 15% (w/v) when NaC1 and KC1 were both absent. Ligation was reduced as the concentration of NaC1 or KC1 was raised in a mixture containing 5 or 15% PEG 6000. With 10% PEG 6000, both cohesive- and blunt-end ligation of this ligase increased at high concentrations of salt (150-200 mM NaC1, or 200-250 mM KC1). Further, with 10% PEG 6000, inter- and intramolecular ligation occurred at low salt concentrations (0-100 mM NaC1, or 0-150 mM KC1); only linear oligomers were formed by intermolecular ligation at the high concentrations.