Hemicatenanes form upon inhibition of DNA replication

Plasmid DNA incubated in interphase Xenopus egg extracts is normally assemb led into chromatin and then into synthetic nuclei which undergo one round o f regulated replication. During a study of restriction endonuclease cut pla smid replication intermediates (Rls) by the Brewer-Fangman 2D gel electroph oresis technique, we have observed the formation of a strong spike of X-sha ped DNA molecules in extracts that otherwise yield very little or no Rls, F ormation of these joint molecules is also efficiently induced in replicatio n-competent extracts upon inhibition of replication fork progression by aph idicolin. Although their electrophoretic properties are quite similar to th ose of Holliday junctions, 2D gels of doubly cut plasmids show that these j unctions can link two plasmid molecules at any pair of DNA sequences, with no regard for sequence homology at the branch points, Neutral-neutral-alkal ine 3D gels show that the junctions only contain single strands of parental size and no recombinant strands. A hemicatenane, in which one strand of a duplex is wound around one strand of another duplex, is the most likely str ucture to account for these observations. The mechanism of formation of the se novel joint DNA molecules and their biological implications are discusse d.