The positioning and dynamics of origins of replication in the budding yeast nucleus.
We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)-tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.
Heun
P
P
Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne, Switzerland.
Laroche
T
T
Raghuraman
M K
MK
Gasser
S M
SM
D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus / Q000235:genetics* / Q000648:ultrastructure; D004261:DNA Replication; D016193:G1 Phase; D016195:G2 Phase; D049452:Green Fluorescent Proteins; D017404:In Situ Hybridization, Fluorescence; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D009685:Nuclear Envelope / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000648:ultrastructure
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins