ORC-DEPENDENT AND ORIGIN-SPECIFIC INITIATION OF DNA-REPLICATION AT DEFINED FOCI IN ISOLATED YEAST NUCLEI

We describe an in vitro replication assay from yeast in which the addi tion of intact nuclei to an S-phase nuclear extract results in the inc orporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesiz ed DNA shifts to a density on CsCl gradients, indicative of semiconser vative replication. Initiation occurs in an origin-specific manner and can be detected in G(1)- or S-phase nuclei, but not in G(2)-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensit ive component necessary to promote replication in G(1)-phase nuclei. R eplication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in tr ans by an extract containing wild-type ORC. The initiation of DNA repl ication in cln-deficient nuclei blocked in G(1) indicates that the ORC -dependent prereplication complex is formed before Start. This represe nts the first nonviral and nonembryonic replication system in which DN A replication initiates in an ORC-dependent and origin-specific manner in vitro.