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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 localization of yeast centromeres and the 2-μm circle. Centromeres cluster close to the spindle pole body in late G1, but do not localize to the extreme nuclear periphery. Diploid GA-1190 cells with the cdc4-3 allele synchronized in late G1 were fixed and subjected to IF/FISH. (a) Four different centromeres, those of ChrVIII, XI, XIII, and XIV, have been detected by FISH and the signals were analyzed for cluster formation and nuclear localization. Centromere probes, green; nuclear pore, red. (Inset) Centromere probes, green; anti–p90 (Spc98, which localizes to the spindle pole body), red; DNA stain (TOTO-3; Molecular Probes), blue. (b) Quantification of the clustering of centromere signals. The criteria for scoring clustering was that the labeled centromeres fall within a circle containing 16% of the nuclear surface at the midsection focal plane (a). (c) Distance-to-edge measurements for the centers of the centromere cluster are displayed in five different nuclear zones (n = 546 signals, 208 nuclei). (d) The early replicating, endogenous 2-μm plasmid clusters in internal regions of the nucleus. Cells were probed for the 2-μm circle (red) and the subtelomeric Y′ element. Nuclear pore, blue. (e) Quantification of the number of 2-μm foci found in 2-D sections of the nuclei. (f) Distance-to-edge measurements (n = 541 signals, 109 nuclei). A χ2 test revealed a significant nonrandom distribution for centromeres (P < 0.05) and a highly significant enrichment of the 2-μm signals at the nuclear interior (P < 0.001). Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
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Figure 6: The localization of yeast centromeres and the 2-μm circle. Centromeres cluster close to the spindle pole body in late G1, but do not localize to the extreme nuclear periphery. Diploid GA-1190 cells with the cdc4-3 allele synchronized in late G1 were fixed and subjected to IF/FISH. (a) Four different centromeres, those of ChrVIII, XI, XIII, and XIV, have been detected by FISH and the signals were analyzed for cluster formation and nuclear localization. Centromere probes, green; nuclear pore, red. (Inset) Centromere probes, green; anti–p90 (Spc98, which localizes to the spindle pole body), red; DNA stain (TOTO-3; Molecular Probes), blue. (b) Quantification of the clustering of centromere signals. The criteria for scoring clustering was that the labeled centromeres fall within a circle containing 16% of the nuclear surface at the midsection focal plane (a). (c) Distance-to-edge measurements for the centers of the centromere cluster are displayed in five different nuclear zones (n = 546 signals, 208 nuclei). (d) The early replicating, endogenous 2-μm plasmid clusters in internal regions of the nucleus. Cells were probed for the 2-μm circle (red) and the subtelomeric Y′ element. Nuclear pore, blue. (e) Quantification of the number of 2-μm foci found in 2-D sections of the nuclei. (f) Distance-to-edge measurements (n = 541 signals, 109 nuclei). A χ2 test revealed a significant nonrandom distribution for centromeres (P < 0.05) and a highly significant enrichment of the 2-μm signals at the nuclear interior (P < 0.001). Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.

Mentions: We next asked whether the characteristic pattern of subnuclear localization of early- and late-firing origins applies to other DNA elements. We have chosen centromeres and the extrachromosomal 2-μm circle, both of which replicate early in S phase and have unusually stable segregation properties (Zakian et al. 1979; McCarroll and Fangman 1988). Centromere organization is of particular interest because FISH studies performed on nuclear spreads have suggested that they cluster close to the spindle pole body (SPB), which itself is embedded in the nuclear membrane in yeast (Jin et al. 1998). Our experiments are performed on diploid cdc4-3 cells blocked in late G1 phase, using four different probes recognizing the centromeres of chromosomes VIII, XI, XIII, and XIV (see Materials and Methods). We clearly observe the clustering of centromeres in a subcompartment of the nucleus, scored here as 16% of the surface area (Fig. 6 a, circle). Moreover, codetection of the CEN-probe FISH with anti–SPB immunofluorescence (anti–Spc98, see Materials and Methods) shows that the clustering occurs around the spindle pole body, forming a ring-like structure (Fig. 6, a and b; see also Jin et al. 2000). However, centromere signals do not coincide with the SPB (Hayashi et al. 1998) and distance-to-edge measurements indicate that the centromeres are enriched in zones 2 and 3, but not zone 1, which is most peripheral (Fig. 6, a and c). This distribution of centromeres is significantly nonrandom (P < 0.05), unlike most genomic early origins.


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 localization of yeast centromeres and the 2-μm circle. Centromeres cluster close to the spindle pole body in late G1, but do not localize to the extreme nuclear periphery. Diploid GA-1190 cells with the cdc4-3 allele synchronized in late G1 were fixed and subjected to IF/FISH. (a) Four different centromeres, those of ChrVIII, XI, XIII, and XIV, have been detected by FISH and the signals were analyzed for cluster formation and nuclear localization. Centromere probes, green; nuclear pore, red. (Inset) Centromere probes, green; anti–p90 (Spc98, which localizes to the spindle pole body), red; DNA stain (TOTO-3; Molecular Probes), blue. (b) Quantification of the clustering of centromere signals. The criteria for scoring clustering was that the labeled centromeres fall within a circle containing 16% of the nuclear surface at the midsection focal plane (a). (c) Distance-to-edge measurements for the centers of the centromere cluster are displayed in five different nuclear zones (n = 546 signals, 208 nuclei). (d) The early replicating, endogenous 2-μm plasmid clusters in internal regions of the nucleus. Cells were probed for the 2-μm circle (red) and the subtelomeric Y′ element. Nuclear pore, blue. (e) Quantification of the number of 2-μm foci found in 2-D sections of the nuclei. (f) Distance-to-edge measurements (n = 541 signals, 109 nuclei). A χ2 test revealed a significant nonrandom distribution for centromeres (P < 0.05) and a highly significant enrichment of the 2-μm signals at the nuclear interior (P < 0.001). Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
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Figure 6: The localization of yeast centromeres and the 2-μm circle. Centromeres cluster close to the spindle pole body in late G1, but do not localize to the extreme nuclear periphery. Diploid GA-1190 cells with the cdc4-3 allele synchronized in late G1 were fixed and subjected to IF/FISH. (a) Four different centromeres, those of ChrVIII, XI, XIII, and XIV, have been detected by FISH and the signals were analyzed for cluster formation and nuclear localization. Centromere probes, green; nuclear pore, red. (Inset) Centromere probes, green; anti–p90 (Spc98, which localizes to the spindle pole body), red; DNA stain (TOTO-3; Molecular Probes), blue. (b) Quantification of the clustering of centromere signals. The criteria for scoring clustering was that the labeled centromeres fall within a circle containing 16% of the nuclear surface at the midsection focal plane (a). (c) Distance-to-edge measurements for the centers of the centromere cluster are displayed in five different nuclear zones (n = 546 signals, 208 nuclei). (d) The early replicating, endogenous 2-μm plasmid clusters in internal regions of the nucleus. Cells were probed for the 2-μm circle (red) and the subtelomeric Y′ element. Nuclear pore, blue. (e) Quantification of the number of 2-μm foci found in 2-D sections of the nuclei. (f) Distance-to-edge measurements (n = 541 signals, 109 nuclei). A χ2 test revealed a significant nonrandom distribution for centromeres (P < 0.05) and a highly significant enrichment of the 2-μm signals at the nuclear interior (P < 0.001). Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
Mentions: We next asked whether the characteristic pattern of subnuclear localization of early- and late-firing origins applies to other DNA elements. We have chosen centromeres and the extrachromosomal 2-μm circle, both of which replicate early in S phase and have unusually stable segregation properties (Zakian et al. 1979; McCarroll and Fangman 1988). Centromere organization is of particular interest because FISH studies performed on nuclear spreads have suggested that they cluster close to the spindle pole body (SPB), which itself is embedded in the nuclear membrane in yeast (Jin et al. 1998). Our experiments are performed on diploid cdc4-3 cells blocked in late G1 phase, using four different probes recognizing the centromeres of chromosomes VIII, XI, XIII, and XIV (see Materials and Methods). We clearly observe the clustering of centromeres in a subcompartment of the nucleus, scored here as 16% of the surface area (Fig. 6 a, circle). Moreover, codetection of the CEN-probe FISH with anti–SPB immunofluorescence (anti–Spc98, see Materials and Methods) shows that the clustering occurs around the spindle pole body, forming a ring-like structure (Fig. 6, a and b; see also Jin et al. 2000). However, centromere signals do not coincide with the SPB (Hayashi et al. 1998) and distance-to-edge measurements indicate that the centromeres are enriched in zones 2 and 3, but not zone 1, which is most peripheral (Fig. 6, a and c). This distribution of centromeres is significantly nonrandom (P < 0.05), unlike most genomic early origins.

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