Structure of the ColE1 DNA molecule before segregation to daughter molecules

Abstract

The segregation of daughter DNA molecules at the end stage of replication of plasmid ColE1 was examined. When circular ColE1 DNA replicates in a cell extract at a high KCl concentration (140 mM), a unique class of molecules accumulates. When the molecule is cleaved by a restriction enzyme that cuts the ColE1 DNA at a single site, an X-shaped molecule in which two linear components are held together around the origin of DNA replication is made. For a large fraction of these molecules, the 5′ end of the leading strand remains at the origin and the 3′ end of the strand is about 30 nucleotides upstream of the origin. The 3′ end of the lagging strand is located at the terH site (17 nucleotides upstream of the origin) and the 5′ end of the strand is a few hundred nucleotides upstream of the terH site. Thus the parental strands of the molecule intertwine with each other only once. When the KCl concentration is lowered to 70 mM, practically all of these molecules are converted to daughter circular monomers or to catenanes consisting of two singly interlocked circular units.