Initiation of DNA replication in Bacillus subtilis: II. Linkage between termini of parental strands and origins of daughter strands

Abstract

Semi-conservative replication, without premature re-initiation, occurred when 5-bromodeoxyuridine was added to thymine-requiring Bacillus subtilis spores which had germinated for three hours in a thymineless medium. When 5-bromodeoxyuridine was added after four hours of thymineless germination, premature re-initiation took place shortly after the first round of replication had begun. Spores were labeled for 30 seconds with 5-[ 3H]bromodeoxyuridine after thymineless germination. The labeled single-stranded DNA was heterogeneous, with molecular weight averaging 3 × 10 6 daltons. The buoyant density of this DNA in alkaline cesium chloride was slightly higher than that of normal thymidine strands. Labeled DNA, chased with non-labeled 5-bromodeoxyuridine, showed different density distribution from that of pulse-labeled strands. Density distributions in alkaline CsCl ranged from the density of a heavy, 5-bromodeoxyuridine-substituted, strand to that of a light normal thymidine strand. The frequencies of various densities were nearly equal. No change in this distribution was observed during prolonged chases if spores germinated for three hours in a thymineless medium were used. However, when spores germinated for four hours without thymine were pulse-labeled and chased until re-initiation began, the density of labeled strands shifted to that of the heavy, fully 5-bromodeoxyuridine-substituted strand. The molecular weights of the chased strands were considerably higher than those of pulse-labeled strands, and were similar to the molecular weight of parental strands. These results indicate that pulse-labeled strands are end-to-end hybrids of heavy 5-bromodeoxyuridine-labeled strands linked to termini of light parental strands. These linkages are stable during the first round of replication but are broken upon initiation of the second round of replication. In consequence we propose that replicating B. subtilis chromosomes are circular, covalently joined between parental strands and newly synthesized strands at the origin of replication. The mechanism of initiation and re-initiation is discussed.