Endo R DpnI restriction of Escherichia coli DNA synthesized in vitro. Evidence that the ends of Okazaki pieces are determined by template deoxynucleotide sequence

Abstract

A restriction enzyme from Diplococcus pneumoniae, Endo R DpnI, cuts methylated DNA on cellophane discs into pieces which are about the same size as Okazaki pieces. DNA was synthesized in vitro on cellophane discs in the presence of β-nicotinamide mononucleotide to prevent joining of Okazaki pieces. This DNA was methylated by the addition of S-adenosyl methionine to the reaction mixture. When Endo R DpnI was used to cut methylated DNA made in vitro in the presence of S-adenosyl methionine and β-nicotinamide mononucleotide, no decrease in sedimentation of the Okazaki pieces was observed. Control experiments demonstrated that Okazaki pieces were methylated in vitro and that Endo R DpnI was capable of cutting double-stranded DNA containing methylated Okazaki pieces, that is, synthesized in β-nicotinamide mononucleotide and S-adenosyl methionine. These results are interpreted to mean that the ends of Okazaki pieces are non-randomly distributed with respect to 6-methyl adenine residues.