Initiation of eukaryotic DNA replication: Origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha

We report that a plasmid replicating in Xenopus egg extracts becomes negati vely supercoiled during replication initiation. Supercoiling requires the i nitiation factor Cdc45, as well as the single-stranded DNA-binding protein RPA, and therefore likely represents origin unwinding. When unwinding is pr evented, Cdc45 binds to chromatin whereas DNA polymerase alpha does not, in dicating that Cdc45, RPA, and DNA polymerase a bind chromatin sequentially at the G1/S transition. Whereas the extent of origin unwinding is normally limited, it increases dramatically when DNA polymerase alpha is inhibited, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork. We discuss the implications of these r esults for the location of replication start sites relative to the prerepli cation complex.