Relaxation complexes of plasmid DNA and protein. II. Characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid ColE1.

Ma Lovett,Dr Helinski

doi:10.1016/s0021-9258(19)40742-4

Abstract

The proteins of the DNA-protein relaxation complex of plasmid ColE1 were labeled with [3H]leucine by growth of ColE1 containing Escherichia coli cells in the presence of this radioactive labeled amino acid. Three major [3H]leucine-labeled proteins are found associated with the supercoiled DNA in the ColE1 relaxation complex. The molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. Induction of relaxation of the supercoiled DNA by treatment of the complex with sodium dodecyl sulfate results in a dissociation of the two smaller proteins from the DNA. The 60,000 protein, however, remains associated specifically with the nicked strand of the open circular DNA. The strand-specific association of this protein with the relaxed DNA resists heat denaturation of the DNA, sedimentation through an alkaline (pH 12.5) sucrose gradient, and centrifugation to equilibrium in an alkaline (pH 12.5) CsCl gradient.

Highlights

ColEl DNA is associated with protein and can be converted from the supercoiled form to the open circular DNA form upon treatment with sodium dodecyl sulfate
The results presented in this report show that the product of sodium dodecyl sulfate-induced relaxation of the ColEl complex contains the 60,000-molecular weight component of the noninduced relaxation complex joined to the nicked strand of
The complete conservation of the strand-specific association of the 60,000 molecular weight protein upon sedimentation through alkaline sucrose, centrifugation to equilibrium in alkaline CsCl, and heat denaturation suggest the existence of a covalent linkage between the protein and the relaxed ColEl DNA

Summary

Introduction

It will be shown that there are three major leucine-labeled proteins associated with supercoiled ColEl DNA in the ColEl relaxation complex, and that following the induction of relaxation with sodium dodecyl sulfate one of the protein components, exhibiting a molecular weight of 60,000, is found in association with the broken strand. ‘The term relaxation complex used in this manuscript refers to a complex of supercoiled (covalently closed circular) DNA plus protein (noncovalently linked to the DNA)

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