Effect of histone H1, poly(ethyleneglycol) and DNA concentration on intermolecular and intramolecular ligation by T4 DNA ligase.
The efficiency of ligation of linear DNA and the relative amounts of intramolecular versus intermolecular ligation may be triggered by a number of additive agents. The results show that it is possible to mimic the effect of poly(ethyleneglycol) 6000 by simply increasing DNA concentration about 15-fold: both the rate and the extent of the reaction are greatly enhanced, and intermolecular ligation is largely favored. However, in this case the stimulation by salts, which occurs in poly(ethyleneglycol) solutions, is not observed; we suggest that salts enhance the hydrophobic interactions between ligase and DNA that take place in the presence of poly(ethyleneglycol). We also show that histone H1, which is involved in the formation of chromatin fibers, is able to stimulate intermolecular ligation by T4 ligase. This effect is more specific than a simple neutralisation of the phosphate groups of the DNA by positive charges of the histone; it still occurs at 125 mM NaCl and in the presence of the four core histones. The implications of the finding concerning the mode of action of histone H1 on DNA are discussed.
Sobczak
J
J
Laboratoire d'Enzymologie des Acides Nucleiques, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Paris, France.
Duguet
M
M
D011088:DNA Ligases / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology; D006657:Histones / Q000494:pharmacology*; D007700:Kinetics; D010957:Plasmids; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011118:Polynucleotide Ligases / Q000378:metabolism*; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology*
D006657:Histones; D011092:Polyethylene Glycols; D004262:DNA Restriction Enzymes; D011088:DNA Ligases; D011118:Polynucleotide Ligases