STUDY OF THE CELL CYCLE-DEPENDENT ASSEMBLY OF THE DNA PRE-REPLICATIONCENTERS IN XENOPUS EGG EXTRACTS

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding pro tein, which assists T-antigen in the assembly of the pre-priming compl ex in the SV40 replication system. By immunodepletion and complementat ion, we have identified RPA as an essential factor for cellular DNA re plication in Xenopus extracts. RPA assembles post-mitotically on the d econdensing chromosomes into numerous subnuclear pre-replication centr es (preRCs) which serve, upon formation of the nuclear membrane, as RC s for the initiation of DNA synthesis. By a variety of experiments inc luding the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assem bly of the preRCs. In contrast, the active cdk2-cyclin A kinase does n ot impede or facilitate the assembly of preRCs. Digestion analysis usi ng the single-strand-specific P1 nuclease as well as competition exper iments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replicati on prior to the unwinding DNA step, but after assembly of preRC and nu clear reconstruction.