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

Heun, Patrick
Raghuraman, M.K.
Published in The journal of cell biology. 2001, vol. 152, no. 2, p. 385-400
Abstract 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.
Keywords Cell cyclePhysiologyCell nucleusGeneticsUltrastructureDNA replicationG1 phaseG2 phaseGreen fluorescent proteinsIn situ hybridizationLuminescent proteinsAnalysisNuclear envelopeRecombinant fusion proteinsReplication originS phaseSaccharomyces cerevisiaeCytology
PMID: 11266454
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HEUN, Patrick et al. The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus. In: The Journal of Cell Biology, 2001, vol. 152, n° 2, p. 385-400. doi: 10.1083/jcb.152.2.385

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Deposited on : 2019-09-13

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