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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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The late replicating plasmid p12 is enriched at the nuclear periphery, but does not colocalize with genomic ARS1412. (a and b) The plasmid p12ARS carries ∼1.5 kb of the core ARS1412 sequence, which fires late in its genomic location but becomes activated early on p12ARS (Friedman et al. 1996). The plasmid p12 bears an additional 17 kb of flanking sequences up- and downstream of the core ARS1412 and maintains a late activation in S phase. Subnuclear localization based on distance-to-edge measurements was quantified for p12ARS and p12, which were probed in diploid cdc4-3 cells that were carrying the appropriate plasmid and were blocked in G1 by a shift to restrictive temperature. Quantification of the FISH signal distribution was performed as described in Fig. 3 a. Numbers are based on two experiments for each plasmid and error bars represent standard deviations. p12ARS (n = 81 signals, 58 nuclei), p12 (n = 219 signals, 143 nuclei). A χ2 test shows that p12 is significantly enriched in the nuclear periphery to P < 0.01, while p12ARS is randomly distributed (P = 0.5). (c and d) Shown are representative confocal images of diploid cdc4-3 cells (GA-1190), blocked in late G1, and labeled with pairs of FISH probes and antinuclear pore (blue). In c, FISH probes recognize the genomic ARS1412 (green) and ARS1413 (red) loci, which colocalize. In d, cells carrying the late replicating plasmid p12 were probed for the plasmid (green) and the genomic ARS1413 (red). The signals were quantified for colocalization and the result is presented in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
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Figure 4: The late replicating plasmid p12 is enriched at the nuclear periphery, but does not colocalize with genomic ARS1412. (a and b) The plasmid p12ARS carries ∼1.5 kb of the core ARS1412 sequence, which fires late in its genomic location but becomes activated early on p12ARS (Friedman et al. 1996). The plasmid p12 bears an additional 17 kb of flanking sequences up- and downstream of the core ARS1412 and maintains a late activation in S phase. Subnuclear localization based on distance-to-edge measurements was quantified for p12ARS and p12, which were probed in diploid cdc4-3 cells that were carrying the appropriate plasmid and were blocked in G1 by a shift to restrictive temperature. Quantification of the FISH signal distribution was performed as described in Fig. 3 a. Numbers are based on two experiments for each plasmid and error bars represent standard deviations. p12ARS (n = 81 signals, 58 nuclei), p12 (n = 219 signals, 143 nuclei). A χ2 test shows that p12 is significantly enriched in the nuclear periphery to P < 0.01, while p12ARS is randomly distributed (P = 0.5). (c and d) Shown are representative confocal images of diploid cdc4-3 cells (GA-1190), blocked in late G1, and labeled with pairs of FISH probes and antinuclear pore (blue). In c, FISH probes recognize the genomic ARS1412 (green) and ARS1413 (red) loci, which colocalize. In d, cells carrying the late replicating plasmid p12 were probed for the plasmid (green) and the genomic ARS1413 (red). The signals were quantified for colocalization and the result is presented in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.

Mentions: Studies to determine elements responsible for the late activation of origins suggest that the flanking DNA plays an important role in establishing and maintaining late initiation (Friedman et al. 1996). Notably, a plasmid bearing only the core of the late-firing origin ARS1412 (p12ARS) is early replicating, while the same plasmid carrying an additional 16 kb of flanking DNA (p12) replicates almost as late as the origin in its genomic location. Based on our previous results, we asked whether these “late DNA elements,” which appear to determine the timing of replication, also determine nuclear localization of the plasmids. Both plasmids are detected by FISH in diploid cdc4-3 cells blocked in late G1 phase. Although the early replicating p12ARS is randomly localized (37 ± 0.5% in zone 1, Fig. 4 a), we again observed an enrichment in peripheral regions of the nucleus for the late-replicating plasmid p12 (49 ± 4.8% in zone 1, Fig. 4 b). This was scored for a large number of signals (n = 300 total) and the χ2 analysis again confirms that the peripheral enrichment of the late-replicating plasmid is highly significant (P < 0.01). These results are consistent with a model in which flanking sequences, or proteins bound to them, are responsible for targeting the late-replicating plasmid to the nuclear periphery.


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)

The late replicating plasmid p12 is enriched at the nuclear periphery, but does not colocalize with genomic ARS1412. (a and b) The plasmid p12ARS carries ∼1.5 kb of the core ARS1412 sequence, which fires late in its genomic location but becomes activated early on p12ARS (Friedman et al. 1996). The plasmid p12 bears an additional 17 kb of flanking sequences up- and downstream of the core ARS1412 and maintains a late activation in S phase. Subnuclear localization based on distance-to-edge measurements was quantified for p12ARS and p12, which were probed in diploid cdc4-3 cells that were carrying the appropriate plasmid and were blocked in G1 by a shift to restrictive temperature. Quantification of the FISH signal distribution was performed as described in Fig. 3 a. Numbers are based on two experiments for each plasmid and error bars represent standard deviations. p12ARS (n = 81 signals, 58 nuclei), p12 (n = 219 signals, 143 nuclei). A χ2 test shows that p12 is significantly enriched in the nuclear periphery to P < 0.01, while p12ARS is randomly distributed (P = 0.5). (c and d) Shown are representative confocal images of diploid cdc4-3 cells (GA-1190), blocked in late G1, and labeled with pairs of FISH probes and antinuclear pore (blue). In c, FISH probes recognize the genomic ARS1412 (green) and ARS1413 (red) loci, which colocalize. In d, cells carrying the late replicating plasmid p12 were probed for the plasmid (green) and the genomic ARS1413 (red). The signals were quantified for colocalization and the result is presented in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
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Figure 4: The late replicating plasmid p12 is enriched at the nuclear periphery, but does not colocalize with genomic ARS1412. (a and b) The plasmid p12ARS carries ∼1.5 kb of the core ARS1412 sequence, which fires late in its genomic location but becomes activated early on p12ARS (Friedman et al. 1996). The plasmid p12 bears an additional 17 kb of flanking sequences up- and downstream of the core ARS1412 and maintains a late activation in S phase. Subnuclear localization based on distance-to-edge measurements was quantified for p12ARS and p12, which were probed in diploid cdc4-3 cells that were carrying the appropriate plasmid and were blocked in G1 by a shift to restrictive temperature. Quantification of the FISH signal distribution was performed as described in Fig. 3 a. Numbers are based on two experiments for each plasmid and error bars represent standard deviations. p12ARS (n = 81 signals, 58 nuclei), p12 (n = 219 signals, 143 nuclei). A χ2 test shows that p12 is significantly enriched in the nuclear periphery to P < 0.01, while p12ARS is randomly distributed (P = 0.5). (c and d) Shown are representative confocal images of diploid cdc4-3 cells (GA-1190), blocked in late G1, and labeled with pairs of FISH probes and antinuclear pore (blue). In c, FISH probes recognize the genomic ARS1412 (green) and ARS1413 (red) loci, which colocalize. In d, cells carrying the late replicating plasmid p12 were probed for the plasmid (green) and the genomic ARS1413 (red). The signals were quantified for colocalization and the result is presented in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
Mentions: Studies to determine elements responsible for the late activation of origins suggest that the flanking DNA plays an important role in establishing and maintaining late initiation (Friedman et al. 1996). Notably, a plasmid bearing only the core of the late-firing origin ARS1412 (p12ARS) is early replicating, while the same plasmid carrying an additional 16 kb of flanking DNA (p12) replicates almost as late as the origin in its genomic location. Based on our previous results, we asked whether these “late DNA elements,” which appear to determine the timing of replication, also determine nuclear localization of the plasmids. Both plasmids are detected by FISH in diploid cdc4-3 cells blocked in late G1 phase. Although the early replicating p12ARS is randomly localized (37 ± 0.5% in zone 1, Fig. 4 a), we again observed an enrichment in peripheral regions of the nucleus for the late-replicating plasmid p12 (49 ± 4.8% in zone 1, Fig. 4 b). This was scored for a large number of signals (n = 300 total) and the χ2 analysis again confirms that the peripheral enrichment of the late-replicating plasmid is highly significant (P < 0.01). These results are consistent with a model in which flanking sequences, or proteins bound to them, are responsible for targeting the late-replicating plasmid to the nuclear periphery.

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