Influence of monovalent cations on the activity of T4 DNA ligase in the presence of polyethylene glycol.
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.
Hayashi
K
K
Nakazawa
M
M
Ishizaki
Y
Y
Obayashi
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D011088:DNA Ligases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011118:Polynucleotide Ligases / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D012964:Sodium / Q000494:pharmacology*; D013604:T-Phages / Q000201:enzymology*; D013696:Temperature
D010710:Phosphates; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D012964:Sodium; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D011118:Polynucleotide Ligases; D011188:Potassium