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© The Rockefeller University Press, 0021-9525/2001//385 $5.00
The Journal of Cell Biology, Volume 152, Number 2, , 2001 385-400

The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

^a Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne, Switzerland
^b Department of Genetics, University of Washington, Seattle, Washington 98195
Swiss Institute for Experimental Cancer Research (ISREC), Chemin des Boveresses 155, CH-1066 Epalinges/Lausanne, Switzerland.41 21 652 693341 21 692 5886

sgasser{at}eliot.unil.ch

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.

Key Words: DNA replication • nuclear organization • in vivo green fluorescent protein imaging • origins of replication • nuclear envelope

© 2001 The Rockefeller University Press

Abbreviations used in this paper: 3-D, three-dimensional; ARS, autonomously replicating sequence; GFP, green fluorescent protein; IF/FISH, immunofluorescence/in situ hybridization.

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