Proteins tightly bound to DNA and DNA‐synthesizing activity of nucleoids from Escherichia coli

Membrane‐attached nucleoids were isolated from E. coli and separated from proteins by 2 m NaCl. Desintegration of such nucleoids by ultrasound and subsequent centrifugation resulted in the formation of two fractions: a sediment (fraction I) and a supernatant (fraction II). The protein:DNA ratio of fraction I was equal to 27 and was different from that to fraction II (2.6). More than 70% of the proteins not dissociating at 2 m NaCl and bound to DNA of both fractions were polypeptides with molecular weights (Mw) of 31,000–23,000 daltons (31–23 Kdal). After pulse labelling of the cells with [ 3 H]‐thymidine, the specific radioactivity of newly synthesized DNA associated with fraction I was shown to be considerably higher than that of fraction II. The analysis of DNA‐synthesizing activities in fractions I and II showed that both nucleoid fractions contained DNA polymerase I. After dissolving the two fractions in 8 m urea – 0.15% sodium dodecylsulphate (SDS) they were chromatographed on hydroxyapatite. DNA‐protein complexes were obtained that did not dissociate at 4 m guanidine. HCI – 5 m urea and 1% SDS. The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA.