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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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In situ hybridization of early- (E) and late- (L) firing origins of replication. (a) Origins for which the timing of replication has been determined by density gradient fractionation (Shirahige et al. 1993; Friedman et al. 1996) were chosen and appropriate FISH probes were prepared as described in Materials and Methods. Early and late origins are indicated in green and red, respectively (see Table ). The Y′ subtelomeric repeat element is not only found at the telomere of chromosome IV, but is at 70–80% of yeast telomeres (Louis et al. 1994). (b–g) IF/FISH has been performed on diploid GA-116 (cdc4-3/cdc4-3) cells that were synchronized in late G1 by a shift to restrictive temperature. Shown are representative examples of confocal images of the mid-cell plane showing nuclei stained for antipore (red) and hybridized with fluorescent probes (green) for the subtelomeric Y′ element, late-activated origins (ARS1412 and ChrIV-210), and early-activated origins (ChrIV-908 and ARS607). (g) Distance-to-edge measurements were performed by categorizing each FISH signal into one of five zones delineated by concentric circles. Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
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Figure 2: In situ hybridization of early- (E) and late- (L) firing origins of replication. (a) Origins for which the timing of replication has been determined by density gradient fractionation (Shirahige et al. 1993; Friedman et al. 1996) were chosen and appropriate FISH probes were prepared as described in Materials and Methods. Early and late origins are indicated in green and red, respectively (see Table ). The Y′ subtelomeric repeat element is not only found at the telomere of chromosome IV, but is at 70–80% of yeast telomeres (Louis et al. 1994). (b–g) IF/FISH has been performed on diploid GA-116 (cdc4-3/cdc4-3) cells that were synchronized in late G1 by a shift to restrictive temperature. Shown are representative examples of confocal images of the mid-cell plane showing nuclei stained for antipore (red) and hybridized with fluorescent probes (green) for the subtelomeric Y′ element, late-activated origins (ARS1412 and ChrIV-210), and early-activated origins (ChrIV-908 and ARS607). (g) Distance-to-edge measurements were performed by categorizing each FISH signal into one of five zones delineated by concentric circles. Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.

Mentions: Short DNA probes were designed to recognize sequences flanking six late-firing origins, six early-firing origins, and the subtelomeric Y′ element that contains a silent origin (Fig. 2 a, and Table ). The regions chosen were based on published information (Shirahige et al. 1993; Friedman et al. 1996, Friedman et al. 1997) and on DNA microarray data that characterized the timing of replication and approximate origin positions on all 16 yeast chromosomes (Raghuraman, M.K., B. Brewer, and W. Fangman, personal communication). FISH probes are obtained by nick-translation of plasmid- or PCR-based DNA templates in the presence of derivatized nucleotides, designed to recognize from 6 to 8 kb of genomic DNA (see Materials and Methods). Diploid cdc4-3 yeast cells (GA-116) were synchronized in late G1 by shifting a culture in an exponential growth phase to restrictive temperature for one generation time (∼105 min). Conditions optimized for the maintenance of a spherical nuclear structure were used to label both nuclear pores and the indicated origins (Fig. 1 a and 2, b–f). Serial confocal microscopy images (z-scans) were performed in each experiment, followed by 3-D reconstruction to evaluate the quality of the nuclear preservation. For each probe, we performed distance-to-edge measurements of the FISH signals on 50–100 nuclei selected on the basis of having a bright undistorted nuclear pore staining that was either round or oval shaped. Typically, the efficiency of in situ hybridization is such that 40% of the cells with intact nuclear pore staining display at least one FISH signal on an equatorial focal section.


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)

In situ hybridization of early- (E) and late- (L) firing origins of replication. (a) Origins for which the timing of replication has been determined by density gradient fractionation (Shirahige et al. 1993; Friedman et al. 1996) were chosen and appropriate FISH probes were prepared as described in Materials and Methods. Early and late origins are indicated in green and red, respectively (see Table ). The Y′ subtelomeric repeat element is not only found at the telomere of chromosome IV, but is at 70–80% of yeast telomeres (Louis et al. 1994). (b–g) IF/FISH has been performed on diploid GA-116 (cdc4-3/cdc4-3) cells that were synchronized in late G1 by a shift to restrictive temperature. Shown are representative examples of confocal images of the mid-cell plane showing nuclei stained for antipore (red) and hybridized with fluorescent probes (green) for the subtelomeric Y′ element, late-activated origins (ARS1412 and ChrIV-210), and early-activated origins (ChrIV-908 and ARS607). (g) Distance-to-edge measurements were performed by categorizing each FISH signal into one of five zones delineated by concentric circles. Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
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Related In: Results  -  Collection

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Figure 2: In situ hybridization of early- (E) and late- (L) firing origins of replication. (a) Origins for which the timing of replication has been determined by density gradient fractionation (Shirahige et al. 1993; Friedman et al. 1996) were chosen and appropriate FISH probes were prepared as described in Materials and Methods. Early and late origins are indicated in green and red, respectively (see Table ). The Y′ subtelomeric repeat element is not only found at the telomere of chromosome IV, but is at 70–80% of yeast telomeres (Louis et al. 1994). (b–g) IF/FISH has been performed on diploid GA-116 (cdc4-3/cdc4-3) cells that were synchronized in late G1 by a shift to restrictive temperature. Shown are representative examples of confocal images of the mid-cell plane showing nuclei stained for antipore (red) and hybridized with fluorescent probes (green) for the subtelomeric Y′ element, late-activated origins (ARS1412 and ChrIV-210), and early-activated origins (ChrIV-908 and ARS607). (g) Distance-to-edge measurements were performed by categorizing each FISH signal into one of five zones delineated by concentric circles. Images were collected on an LSM 410 confocal microscope. Scale bars: 2 μm.
Mentions: Short DNA probes were designed to recognize sequences flanking six late-firing origins, six early-firing origins, and the subtelomeric Y′ element that contains a silent origin (Fig. 2 a, and Table ). The regions chosen were based on published information (Shirahige et al. 1993; Friedman et al. 1996, Friedman et al. 1997) and on DNA microarray data that characterized the timing of replication and approximate origin positions on all 16 yeast chromosomes (Raghuraman, M.K., B. Brewer, and W. Fangman, personal communication). FISH probes are obtained by nick-translation of plasmid- or PCR-based DNA templates in the presence of derivatized nucleotides, designed to recognize from 6 to 8 kb of genomic DNA (see Materials and Methods). Diploid cdc4-3 yeast cells (GA-116) were synchronized in late G1 by shifting a culture in an exponential growth phase to restrictive temperature for one generation time (∼105 min). Conditions optimized for the maintenance of a spherical nuclear structure were used to label both nuclear pores and the indicated origins (Fig. 1 a and 2, b–f). Serial confocal microscopy images (z-scans) were performed in each experiment, followed by 3-D reconstruction to evaluate the quality of the nuclear preservation. For each probe, we performed distance-to-edge measurements of the FISH signals on 50–100 nuclei selected on the basis of having a bright undistorted nuclear pore staining that was either round or oval shaped. Typically, the efficiency of in situ hybridization is such that 40% of the cells with intact nuclear pore staining display at least one FISH signal on an equatorial focal section.

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