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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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Dynamics of origins in vivo: a nontelomeric late origin becomes increasingly nonperipheral in S and G2 phases. Origin localization has been followed in living cells by the use of the lac operator/lac repressor system. A repeat of 256 lac operator sites has been integrated in wild-type budding yeast cells (GA-1320), either at the late origin cluster on chromosome XIV, ∼10 kb upstream of ARS1413 (a), or ∼3 kb upstream of the early activated origin ChrIV-908 (d, see Materials and Methods). These cells also carry a GFP-Nup49p fusion protein to monitor the nuclear periphery. Log phase cells (<106 cells/ml) were concentrated by a short centrifugation and transferred to agar lacking histidine on a microscope slide. Transmission and fluorescence microscopy pictures were taken on an Olympus microscope coupled to the TillVision® imaging system. In nuclei where the GFP-tagged origin and the nuclear equator coincide in the same focal plane, origin position from the midpore signal was determined using the line profile tool of LSM510 Confocal Software. Distances were classified peripheral or internal using the 50% methods described in Fig. 3 b and 7. Cell-cycle stages were based on both cellular and nuclear shape and categorized as G1, early S, mid S, lateS/G2, and M phase (see Materials and Methods). Shown are results for the late-origin cluster on chromosome XIV (c; n = 161 nuclei) and the early activated origin ChrIV-908 (f; n = 165 nuclei). (b and e) One cell for each stage is shown as merged transmission/fluorescence images (top) and the fluorescence alone (bottom). A χ2 test demonstrates no significant difference from random localization for any origin at any cell-cycle phase, except for the late origin cluster on chromosome XIV in G1 (P < 0.001). Bar = 2 μm.
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Figure 8: Dynamics of origins in vivo: a nontelomeric late origin becomes increasingly nonperipheral in S and G2 phases. Origin localization has been followed in living cells by the use of the lac operator/lac repressor system. A repeat of 256 lac operator sites has been integrated in wild-type budding yeast cells (GA-1320), either at the late origin cluster on chromosome XIV, ∼10 kb upstream of ARS1413 (a), or ∼3 kb upstream of the early activated origin ChrIV-908 (d, see Materials and Methods). These cells also carry a GFP-Nup49p fusion protein to monitor the nuclear periphery. Log phase cells (<106 cells/ml) were concentrated by a short centrifugation and transferred to agar lacking histidine on a microscope slide. Transmission and fluorescence microscopy pictures were taken on an Olympus microscope coupled to the TillVision® imaging system. In nuclei where the GFP-tagged origin and the nuclear equator coincide in the same focal plane, origin position from the midpore signal was determined using the line profile tool of LSM510 Confocal Software. Distances were classified peripheral or internal using the 50% methods described in Fig. 3 b and 7. Cell-cycle stages were based on both cellular and nuclear shape and categorized as G1, early S, mid S, lateS/G2, and M phase (see Materials and Methods). Shown are results for the late-origin cluster on chromosome XIV (c; n = 161 nuclei) and the early activated origin ChrIV-908 (f; n = 165 nuclei). (b and e) One cell for each stage is shown as merged transmission/fluorescence images (top) and the fluorescence alone (bottom). A χ2 test demonstrates no significant difference from random localization for any origin at any cell-cycle phase, except for the late origin cluster on chromosome XIV in G1 (P < 0.001). Bar = 2 μm.

Mentions: The FISH analysis of the recombinase-sensitive telomere-proximal origin suggests that it can diffuse rapidly from Tel V-R upon excision. To confirm this in living cells, we have tagged origins fluorescently to follow their localization in vivo, making use of a lac operator/GFP-lac repressor system (Robinett et al. 1996). A repeat of 256 lac operator sites has been integrated in haploid wild-type cells (GA-1320) at the late origin cluster on chromosome XIV adjacent to ARS1413, or next to the early-activated origin on chromosome IV, 908 kb away from the left telomere (Fig. 8, a and b). These cells carry both a GFP-tagged copy of the nuclear pore protein Nup49p and the GFP-lac repressor fusion protein. The binding of the lac repressor to the multimerized lac sites results in a strong signal that can easily be distinguished from the weaker GFP-nuclear envelope fluorescence, allowing us to monitor the relative position of the tagged origins with respect to the nuclear periphery. Control experiments confirm that the GFP tagging does not significantly alter the timing of replication of the modified region (F. Neumann, personal communication).


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)

Dynamics of origins in vivo: a nontelomeric late origin becomes increasingly nonperipheral in S and G2 phases. Origin localization has been followed in living cells by the use of the lac operator/lac repressor system. A repeat of 256 lac operator sites has been integrated in wild-type budding yeast cells (GA-1320), either at the late origin cluster on chromosome XIV, ∼10 kb upstream of ARS1413 (a), or ∼3 kb upstream of the early activated origin ChrIV-908 (d, see Materials and Methods). These cells also carry a GFP-Nup49p fusion protein to monitor the nuclear periphery. Log phase cells (<106 cells/ml) were concentrated by a short centrifugation and transferred to agar lacking histidine on a microscope slide. Transmission and fluorescence microscopy pictures were taken on an Olympus microscope coupled to the TillVision® imaging system. In nuclei where the GFP-tagged origin and the nuclear equator coincide in the same focal plane, origin position from the midpore signal was determined using the line profile tool of LSM510 Confocal Software. Distances were classified peripheral or internal using the 50% methods described in Fig. 3 b and 7. Cell-cycle stages were based on both cellular and nuclear shape and categorized as G1, early S, mid S, lateS/G2, and M phase (see Materials and Methods). Shown are results for the late-origin cluster on chromosome XIV (c; n = 161 nuclei) and the early activated origin ChrIV-908 (f; n = 165 nuclei). (b and e) One cell for each stage is shown as merged transmission/fluorescence images (top) and the fluorescence alone (bottom). A χ2 test demonstrates no significant difference from random localization for any origin at any cell-cycle phase, except for the late origin cluster on chromosome XIV in G1 (P < 0.001). Bar = 2 μm.
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Figure 8: Dynamics of origins in vivo: a nontelomeric late origin becomes increasingly nonperipheral in S and G2 phases. Origin localization has been followed in living cells by the use of the lac operator/lac repressor system. A repeat of 256 lac operator sites has been integrated in wild-type budding yeast cells (GA-1320), either at the late origin cluster on chromosome XIV, ∼10 kb upstream of ARS1413 (a), or ∼3 kb upstream of the early activated origin ChrIV-908 (d, see Materials and Methods). These cells also carry a GFP-Nup49p fusion protein to monitor the nuclear periphery. Log phase cells (<106 cells/ml) were concentrated by a short centrifugation and transferred to agar lacking histidine on a microscope slide. Transmission and fluorescence microscopy pictures were taken on an Olympus microscope coupled to the TillVision® imaging system. In nuclei where the GFP-tagged origin and the nuclear equator coincide in the same focal plane, origin position from the midpore signal was determined using the line profile tool of LSM510 Confocal Software. Distances were classified peripheral or internal using the 50% methods described in Fig. 3 b and 7. Cell-cycle stages were based on both cellular and nuclear shape and categorized as G1, early S, mid S, lateS/G2, and M phase (see Materials and Methods). Shown are results for the late-origin cluster on chromosome XIV (c; n = 161 nuclei) and the early activated origin ChrIV-908 (f; n = 165 nuclei). (b and e) One cell for each stage is shown as merged transmission/fluorescence images (top) and the fluorescence alone (bottom). A χ2 test demonstrates no significant difference from random localization for any origin at any cell-cycle phase, except for the late origin cluster on chromosome XIV in G1 (P < 0.001). Bar = 2 μm.
Mentions: The FISH analysis of the recombinase-sensitive telomere-proximal origin suggests that it can diffuse rapidly from Tel V-R upon excision. To confirm this in living cells, we have tagged origins fluorescently to follow their localization in vivo, making use of a lac operator/GFP-lac repressor system (Robinett et al. 1996). A repeat of 256 lac operator sites has been integrated in haploid wild-type cells (GA-1320) at the late origin cluster on chromosome XIV adjacent to ARS1413, or next to the early-activated origin on chromosome IV, 908 kb away from the left telomere (Fig. 8, a and b). These cells carry both a GFP-tagged copy of the nuclear pore protein Nup49p and the GFP-lac repressor fusion protein. The binding of the lac repressor to the multimerized lac sites results in a strong signal that can easily be distinguished from the weaker GFP-nuclear envelope fluorescence, allowing us to monitor the relative position of the tagged origins with respect to the nuclear periphery. Control experiments confirm that the GFP tagging does not significantly alter the timing of replication of the modified region (F. Neumann, personal communication).

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
Related in: MedlinePlus