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 addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.
Pasero
P
P
Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne.
Braguglia
D
D
Gasser
S M
SM
D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000302:isolation & purification; D016193:G1 Phase; D018741:Replication Origin; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal