A protein covalently bound to ColEl DNA

Abstract

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein‐denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open‐circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36‐kDa protein which, we believe, is covalently bound to ColEl DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane‐treated‐trypsin and the DNA‐peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE‐Sephadex chromatography. The only 5′‐dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O‐Phosphoserine was found in acid hydrolysates of the DNA‐peptide(s). We suggest that in the relaxation event the 36‐kDa protein becomes covalently linked to ColEl DNA via a phosphodiester bond between dC and the serine residue. Copyright © 1988, Wiley Blackwell. All rights reserved