Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis.

By the use of a restriction enzyme digestion of gently lysed E. coli or B. subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient. Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed. Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques. The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates. In B. subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker.