Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.
We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.
D001412:Bacillus subtilis; D001435:Bacteriophages / Q000032:analysis* / Q000254:growth & development; D002499:Centrifugation, Density Gradient; D004261:DNA Replication; D004277:DNA, Single-Stranded; D004279:DNA, Viral / Q000096:biosynthesis; D008854:Microscopy, Electron; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral