Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers

Abstract

We have identified a new class of SV40 replicative intermediates which consists of a least 20 discrete DNA species. All members of this class are catenated dimers, two circular molecules of SV40 duplex DNA linked topologically by one or more intertwining events. Most of these molecules are linked by several intertwining events, and the range of linkage states observed runs from L = 1 to L = 10. A catenated dimer with a given linkage state is assigned to one of three distinct families (A, B or C) depending on the open or covalently closed nature of its two circular components: in form A catenated dimers, both circles are nicked or gapped; in form B, one of the circles is supercoiled; and in form C, both circles are supercoiled. Members of all three of these families are found in SV40 chromatin pulse-labeled with ³H-thymidine, and together they comprise 10–20% of the total replicative form SV40 DNA, appearing as a discrete series of electrophoretically resolved bands superimposed upon a continuous smear of growing cairns structures. The distribution of linkage states varies between the families, A being the most intertwined and C the least intertwined. Upon a chase with cold thymidine, label is lost rapidly from all these catenated DNA species. We suggest that the sequence A → B → C → mature monomeric supercoiled SV40 DNA represents the final stages of SV40 replication, and that a special enzyme activity exists in vivo to uncatenate the SV40 daughter chromosomes.

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