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

Abstract

Membrane-attached nucleoids were isolated from E. coli and separated from proteins by 2 M NaCl. Disintegration 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 [3H]-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 X HCl - 5 M urea and 1% SDS. The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA.