Involvement of topoisomerases in the initiation of simian virus 40 minichromosome replication

Topoisomerases provide the unlinking activity necessary for replication for k movement during DNA replication. It is uncertain, however, whether topois omerases are also required for the initiation of replication. To investigat e this point, we have performed pulse-chase experiments with SV40 minichrom osomes as template to distinguish between the initiation and the elongation of replication, Using an unfractionated cytosolic extract as a source of r eplication functions, we found that the addition of topoisomerases at the i nitiation step significantly increased the number of active chromatin templ ates, whereas addition of topoisomerases at the elongation step had only mi nor effects. Minichromosomes with an extended chromatin structure as well a s protein-free DNA required less topoisomerase for effective replication in itiation. We could exclude the possibility that topoisomerases enhance the origin binding of T antigen, the SV40 replication initiator, and propose in stead that the arrangement of nucleosomes influences the diffusion of super coils during initial DNA unwinding. Efficient initiation therefore requires a high local concentration of topoisomerases to relax the torsional stress .