Limits...
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.

Show MeSH

Related in: MedlinePlus

Once the late timing is established, ARS501 requires neither telomere proximity nor a peripheral position to remain late firing. Haploid budding yeast cells with the temperature-sensitive cdc7-1 allele and the excisable 30-kb cassette bearing ARS501 (GA-1205; Raghuraman et al. 1997) were blocked with nocodazole at G2/M (c and e) or with α factor at Start in G1 (b and d). The site-specific recombinase was induced by addition of galactose. After a 4-h incubation, cells were released into glucose-containing medium at 37°C, which represses the recombinase and synchronizes cells at the G1/S boundary (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence (blue), FISH with ARS501 (red), and Tel V-R (green). In a, we show a scheme of the FISH probes recognizing 7 kb on the excisable ARS501 circle (red) and an adjacent 5-kb probe that is 14 kb closer to Tel V-R (green). The 30-kb ARS501 cassette is flanked by recombination sites specifically recognized by the R recombinase from Z. rouxii (Raghuraman et al. 1997). (b–e) Codetection of the two FISH probes has been performed as described above and representative confocal images of equatorial sections through hybridized nuclei are shown. In cells with preserved nuclear pore staining, subnuclear localization of the 30-kb ARS501 cassette was quantified for its position relative to either the telomere proximal locus on chromosome V or to the nuclear periphery using the line profile tool of LSM510 Confocal Software. Distance measurements between the maxima of two signals were categorized in groups from 0–99 nm = 100, 100–199 = 200 etc., and expressed in bar graphs in f (nnonexcised; excised = 42, 37) and g (nnonexcised; excised = 24, 22). The general position of the ARS501 cassette within the nucleus was analyzed by computing the ratio between the minimal distance from the pore of the FISH signal and the radius of the nucleus. Values <0.29 were scored as peripheral as in Fig. 3 b (see Materials and Methods). Percentages of peripheral (dark grey) or internal (light grey) localization are presented as bar graphs. Peripheral localization is expressed in percent in f (nnonexcised; excised = 48, 66) and g (nnon-excised; excised = 57, 48), insets. Analysis using a standard χ2 test revealed that the localization of ARS501 is significantly nonrandom before excision (P < 0.001), but not after (P = 0.35). Images were collected on an LSM 510 confocal microscope. Scale bar: 2 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199623&req=5

Figure 7: Once the late timing is established, ARS501 requires neither telomere proximity nor a peripheral position to remain late firing. Haploid budding yeast cells with the temperature-sensitive cdc7-1 allele and the excisable 30-kb cassette bearing ARS501 (GA-1205; Raghuraman et al. 1997) were blocked with nocodazole at G2/M (c and e) or with α factor at Start in G1 (b and d). The site-specific recombinase was induced by addition of galactose. After a 4-h incubation, cells were released into glucose-containing medium at 37°C, which represses the recombinase and synchronizes cells at the G1/S boundary (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence (blue), FISH with ARS501 (red), and Tel V-R (green). In a, we show a scheme of the FISH probes recognizing 7 kb on the excisable ARS501 circle (red) and an adjacent 5-kb probe that is 14 kb closer to Tel V-R (green). The 30-kb ARS501 cassette is flanked by recombination sites specifically recognized by the R recombinase from Z. rouxii (Raghuraman et al. 1997). (b–e) Codetection of the two FISH probes has been performed as described above and representative confocal images of equatorial sections through hybridized nuclei are shown. In cells with preserved nuclear pore staining, subnuclear localization of the 30-kb ARS501 cassette was quantified for its position relative to either the telomere proximal locus on chromosome V or to the nuclear periphery using the line profile tool of LSM510 Confocal Software. Distance measurements between the maxima of two signals were categorized in groups from 0–99 nm = 100, 100–199 = 200 etc., and expressed in bar graphs in f (nnonexcised; excised = 42, 37) and g (nnonexcised; excised = 24, 22). The general position of the ARS501 cassette within the nucleus was analyzed by computing the ratio between the minimal distance from the pore of the FISH signal and the radius of the nucleus. Values <0.29 were scored as peripheral as in Fig. 3 b (see Materials and Methods). Percentages of peripheral (dark grey) or internal (light grey) localization are presented as bar graphs. Peripheral localization is expressed in percent in f (nnonexcised; excised = 48, 66) and g (nnon-excised; excised = 57, 48), insets. Analysis using a standard χ2 test revealed that the localization of ARS501 is significantly nonrandom before excision (P < 0.001), but not after (P = 0.35). Images were collected on an LSM 510 confocal microscope. Scale bar: 2 μm.

Mentions: The yeast strain used for this study carries the temperature-sensitive cdc7-1 allele and direct repeats flanking ARS501 (GA-1205), enabling the excision of a 30-kb circle upon induction of a site-specific recombinase and subsequent synchronization immediately before initiation of DNA replication. Cells were first arrested either at G2/M with nocodazole or at Start in G1 with the pheromone α factor, and the recombinase was induced by the addition of galactose. After a 4-h incubation, release from the arrest points was achieved by transferring cells to inhibitor-free glucose-containing medium at 37°C. This results in two populations of cells synchronized just before initiation of DNA replication: one carries an ARS501 circle excised in midmitosis and the other excised in mid-G1 (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence and FISH with probes recognizing either ARS501 (red) or a nonexcised 5-kb sequence 14-kb closer to the right telomere of chromosome V (Fig. 7 a, green).


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)

Once the late timing is established, ARS501 requires neither telomere proximity nor a peripheral position to remain late firing. Haploid budding yeast cells with the temperature-sensitive cdc7-1 allele and the excisable 30-kb cassette bearing ARS501 (GA-1205; Raghuraman et al. 1997) were blocked with nocodazole at G2/M (c and e) or with α factor at Start in G1 (b and d). The site-specific recombinase was induced by addition of galactose. After a 4-h incubation, cells were released into glucose-containing medium at 37°C, which represses the recombinase and synchronizes cells at the G1/S boundary (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence (blue), FISH with ARS501 (red), and Tel V-R (green). In a, we show a scheme of the FISH probes recognizing 7 kb on the excisable ARS501 circle (red) and an adjacent 5-kb probe that is 14 kb closer to Tel V-R (green). The 30-kb ARS501 cassette is flanked by recombination sites specifically recognized by the R recombinase from Z. rouxii (Raghuraman et al. 1997). (b–e) Codetection of the two FISH probes has been performed as described above and representative confocal images of equatorial sections through hybridized nuclei are shown. In cells with preserved nuclear pore staining, subnuclear localization of the 30-kb ARS501 cassette was quantified for its position relative to either the telomere proximal locus on chromosome V or to the nuclear periphery using the line profile tool of LSM510 Confocal Software. Distance measurements between the maxima of two signals were categorized in groups from 0–99 nm = 100, 100–199 = 200 etc., and expressed in bar graphs in f (nnonexcised; excised = 42, 37) and g (nnonexcised; excised = 24, 22). The general position of the ARS501 cassette within the nucleus was analyzed by computing the ratio between the minimal distance from the pore of the FISH signal and the radius of the nucleus. Values <0.29 were scored as peripheral as in Fig. 3 b (see Materials and Methods). Percentages of peripheral (dark grey) or internal (light grey) localization are presented as bar graphs. Peripheral localization is expressed in percent in f (nnonexcised; excised = 48, 66) and g (nnon-excised; excised = 57, 48), insets. Analysis using a standard χ2 test revealed that the localization of ARS501 is significantly nonrandom before excision (P < 0.001), but not after (P = 0.35). Images were collected on an LSM 510 confocal microscope. Scale bar: 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Once the late timing is established, ARS501 requires neither telomere proximity nor a peripheral position to remain late firing. Haploid budding yeast cells with the temperature-sensitive cdc7-1 allele and the excisable 30-kb cassette bearing ARS501 (GA-1205; Raghuraman et al. 1997) were blocked with nocodazole at G2/M (c and e) or with α factor at Start in G1 (b and d). The site-specific recombinase was induced by addition of galactose. After a 4-h incubation, cells were released into glucose-containing medium at 37°C, which represses the recombinase and synchronizes cells at the G1/S boundary (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence (blue), FISH with ARS501 (red), and Tel V-R (green). In a, we show a scheme of the FISH probes recognizing 7 kb on the excisable ARS501 circle (red) and an adjacent 5-kb probe that is 14 kb closer to Tel V-R (green). The 30-kb ARS501 cassette is flanked by recombination sites specifically recognized by the R recombinase from Z. rouxii (Raghuraman et al. 1997). (b–e) Codetection of the two FISH probes has been performed as described above and representative confocal images of equatorial sections through hybridized nuclei are shown. In cells with preserved nuclear pore staining, subnuclear localization of the 30-kb ARS501 cassette was quantified for its position relative to either the telomere proximal locus on chromosome V or to the nuclear periphery using the line profile tool of LSM510 Confocal Software. Distance measurements between the maxima of two signals were categorized in groups from 0–99 nm = 100, 100–199 = 200 etc., and expressed in bar graphs in f (nnonexcised; excised = 42, 37) and g (nnonexcised; excised = 24, 22). The general position of the ARS501 cassette within the nucleus was analyzed by computing the ratio between the minimal distance from the pore of the FISH signal and the radius of the nucleus. Values <0.29 were scored as peripheral as in Fig. 3 b (see Materials and Methods). Percentages of peripheral (dark grey) or internal (light grey) localization are presented as bar graphs. Peripheral localization is expressed in percent in f (nnonexcised; excised = 48, 66) and g (nnon-excised; excised = 57, 48), insets. Analysis using a standard χ2 test revealed that the localization of ARS501 is significantly nonrandom before excision (P < 0.001), but not after (P = 0.35). Images were collected on an LSM 510 confocal microscope. Scale bar: 2 μm.
Mentions: The yeast strain used for this study carries the temperature-sensitive cdc7-1 allele and direct repeats flanking ARS501 (GA-1205), enabling the excision of a 30-kb circle upon induction of a site-specific recombinase and subsequent synchronization immediately before initiation of DNA replication. Cells were first arrested either at G2/M with nocodazole or at Start in G1 with the pheromone α factor, and the recombinase was induced by the addition of galactose. After a 4-h incubation, release from the arrest points was achieved by transferring cells to inhibitor-free glucose-containing medium at 37°C. This results in two populations of cells synchronized just before initiation of DNA replication: one carries an ARS501 circle excised in midmitosis and the other excised in mid-G1 (see Materials and Methods). Cells were fixed and subjected to nuclear pore immunofluorescence and FISH with probes recognizing either ARS501 (red) or a nonexcised 5-kb sequence 14-kb closer to the right telomere of chromosome V (Fig. 7 a, green).

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