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The positioning and dynamics of origins of replication in the budding yeast nucleus.

Heun P, Laroche T, Raghuraman MK, Gasser SM - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne, Switzerland.

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.

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This model proposes that DNA elements flanking late firing origins promote association with the nuclear periphery in early G1 phase, an event necessary for the establishment of a late firing chromatin state. Our data suggest that it is not necessary to maintain perinuclear positioning to ensure that the origin fires late in S phase. Once formed, late chromatin may retard initiation by rendering the pre–regulatory complex less accessible to specific factors required for initiation, such as Cdc45p, Polα-primase, or either of the regulatory kinases Clb5/Cdc28 or Dbf4/Cdc7. The initiation of replication at early origins may also send signals through factors such as Rad53p to help repress initiation at late-activated origins.
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Figure 9: This model proposes that DNA elements flanking late firing origins promote association with the nuclear periphery in early G1 phase, an event necessary for the establishment of a late firing chromatin state. Our data suggest that it is not necessary to maintain perinuclear positioning to ensure that the origin fires late in S phase. Once formed, late chromatin may retard initiation by rendering the pre–regulatory complex less accessible to specific factors required for initiation, such as Cdc45p, Polα-primase, or either of the regulatory kinases Clb5/Cdc28 or Dbf4/Cdc7. The initiation of replication at early origins may also send signals through factors such as Rad53p to help repress initiation at late-activated origins.

Mentions: Taken together, these results suggest a model in which the necessary prerequisites for late firing would be both contact with the nuclear periphery in G1 and the presence of “late determining DNA elements.” These would either help target the domain to the nuclear periphery in early G1, or allow assembly of a structure capable of retarding the initiation event, or both (Fig. 9). Once established, the modified late chromatin structure appears to be autonomous and mobile. Our data further suggest that a peripheral localization alone is not sufficient to confer delayed initiation, since early replicating origins are found with equal efficiency at internal sites as at the nuclear periphery using both FISH and GFP fluorescence.


The positioning and dynamics of origins of replication in the budding yeast nucleus.

Heun P, Laroche T, Raghuraman MK, Gasser SM - J. Cell Biol. (2001)

This model proposes that DNA elements flanking late firing origins promote association with the nuclear periphery in early G1 phase, an event necessary for the establishment of a late firing chromatin state. Our data suggest that it is not necessary to maintain perinuclear positioning to ensure that the origin fires late in S phase. Once formed, late chromatin may retard initiation by rendering the pre–regulatory complex less accessible to specific factors required for initiation, such as Cdc45p, Polα-primase, or either of the regulatory kinases Clb5/Cdc28 or Dbf4/Cdc7. The initiation of replication at early origins may also send signals through factors such as Rad53p to help repress initiation at late-activated origins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199623&req=5

Figure 9: This model proposes that DNA elements flanking late firing origins promote association with the nuclear periphery in early G1 phase, an event necessary for the establishment of a late firing chromatin state. Our data suggest that it is not necessary to maintain perinuclear positioning to ensure that the origin fires late in S phase. Once formed, late chromatin may retard initiation by rendering the pre–regulatory complex less accessible to specific factors required for initiation, such as Cdc45p, Polα-primase, or either of the regulatory kinases Clb5/Cdc28 or Dbf4/Cdc7. The initiation of replication at early origins may also send signals through factors such as Rad53p to help repress initiation at late-activated origins.
Mentions: Taken together, these results suggest a model in which the necessary prerequisites for late firing would be both contact with the nuclear periphery in G1 and the presence of “late determining DNA elements.” These would either help target the domain to the nuclear periphery in early G1, or allow assembly of a structure capable of retarding the initiation event, or both (Fig. 9). Once established, the modified late chromatin structure appears to be autonomous and mobile. Our data further suggest that a peripheral localization alone is not sufficient to confer delayed initiation, since early replicating origins are found with equal efficiency at internal sites as at the nuclear periphery using both FISH and GFP fluorescence.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges/Lausanne, Switzerland.

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.

Show MeSH