Discontinuous duplication of both strands of virus 2C DNA.

Abstract

When Bacillus subtilis infected with virus 2C was labeled with short pulses of tritiated uracil (a specific precursor of the hydroxymethyluracil replacing thymine in 2C chromosome) and the cell lysate was fractionated in alkaline sucrose gradients, most of the radioactivity was found within a lowmolecular-weight peak of DNA (“Okazaki pieces”) of about 2×106 daltons. After longer pulses, segments of large size and full strands were the main labeled products. A shift from low to high-molecular-weight nascent DNA took place slowly during the eclipse phase, and rapidly during the maturation phase. This suggested a lagging of the ligase behind the polymerase at early stages. During the entire replication cycle, pulse-labeled DNA was present within the replication fork in its native form with no involvement of proteins. Furthermore, the amount of lowmolecular-weight pulse-labeled DNA hybridizing with the L strand was higher than that annealing with the H strand. To explain this finding it is suggested that the polymerization or the ligation of small segments is faster for one strand than for the other. Finally, the DNA fraction which is resistant to single-strand specific nuclease was about 10% when alkali-denatured Okazaki pieces were neutralized at 4C and was increased about five-fold upon selfannealing. It is concluded that both strands of 2C DNA duplicate in a discontinuous fashion through the formation of Okazaki pieces, which are held by hydrogen bonds initially and subsequently by phosphodiester linkages. It is proposed also that ligase action is faster for the L strand than for the H strand, and that this phenomenon might be related to both the polarity of chain growth and the asymmetry of transcription of 2C chromosome.