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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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Late replication origins are enriched near the nuclear periphery. FISH signals were quantified on equatorial confocal sections of diploid strain GA-116, for ARS1412, ChrIV-908, and Y′-containing telomeres (Fig. 2b, Fig. c, and Fig. e) relative to the zones indicated in a–c. ARS1412 and the subtelomeric Y′ elements are late replicating, while ChrIV-908 is early. (a) Distance to edge is measured by the use of five zones, and data are represented in a bar graph as a percentage of signals per zone, each of which has a different area. The line corresponds to theoretical values for randomly distributed signals and is proportional to the surface area of each zone. From left to right (in brackets: n = signals, nuclei): ARS1412 (106, 76), ChrIV-908 (83, 64), and Y′ (264, 66). Each experiment has been performed at least twice and error bars represent standard deviations. Analysis using a standard χ2 test reveals a statistically significant nonrandom localization for ARS1412 (P < 0.05) and Y′ FISH probes (P < 0.001), but not for ChrIV-908 (P = 0.1). (b) The nuclei are divided into two zones of equal surface area: peripheral and internal. FISH signal localization was quantified on a computer graphic representation of 2-D confocal images in which the nuclear pore staining was enlarged to create a peripheral zone that comprises 50% of the original nuclear surface. FISH signals found within this zone were scored as peripheral. ARS1412 (43, 42), ChrIV-908 (59, 44), and Y′ (149, 44). ARS1412 and Y′ signals were scored using nuclei from multiple experiments. (c) 3-D reconstituted nuclei (as in Fig. 1 a) are divided in two spaces of similar volume and the frequency of FISH signals is scored. ARS1412 (5, 5), ChrIV-908 (10, 5), and Y′ (22, 7). (d) The localization of six late-activated (dark columns) and six early-activated (light columns) origins has been quantified by distance-to-edge measurements as described in a. For each zone, the theoretical random signal distribution, based on surface area, has been subtracted from the corresponding signal frequency. A value of 0 corresponds to random distribution of signals; numbers > and < 0 indicate enrichment and depletion, respectively. Columns from left to right (brackets: n = signals, nuclei): ARS1412 (106, 76), ARS1413 (143, 94), ChrIV-210 (112, 78), ChrIV-257 (111, 77), ARS603 (64, 54), ChrX-305 (189, 123) ARS1 (43, 32), ChrIV-908 (83, 64), ChrIV-1153 (123, 86), ARS606 (83, 66), ARS607 (47, 37), and ChrX-613 (81, 63). A χ2 test demonstrates that four out of six late origins and none of the early origin FISH signals differ significantly from a random distribution to P < 0.05. When taken together, the localization of the pooled late origins in zone 1 is significantly nonrandom and is significantly different from the localization of the pooled early origins (P < 0.001).
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Figure 3: Late replication origins are enriched near the nuclear periphery. FISH signals were quantified on equatorial confocal sections of diploid strain GA-116, for ARS1412, ChrIV-908, and Y′-containing telomeres (Fig. 2b, Fig. c, and Fig. e) relative to the zones indicated in a–c. ARS1412 and the subtelomeric Y′ elements are late replicating, while ChrIV-908 is early. (a) Distance to edge is measured by the use of five zones, and data are represented in a bar graph as a percentage of signals per zone, each of which has a different area. The line corresponds to theoretical values for randomly distributed signals and is proportional to the surface area of each zone. From left to right (in brackets: n = signals, nuclei): ARS1412 (106, 76), ChrIV-908 (83, 64), and Y′ (264, 66). Each experiment has been performed at least twice and error bars represent standard deviations. Analysis using a standard χ2 test reveals a statistically significant nonrandom localization for ARS1412 (P < 0.05) and Y′ FISH probes (P < 0.001), but not for ChrIV-908 (P = 0.1). (b) The nuclei are divided into two zones of equal surface area: peripheral and internal. FISH signal localization was quantified on a computer graphic representation of 2-D confocal images in which the nuclear pore staining was enlarged to create a peripheral zone that comprises 50% of the original nuclear surface. FISH signals found within this zone were scored as peripheral. ARS1412 (43, 42), ChrIV-908 (59, 44), and Y′ (149, 44). ARS1412 and Y′ signals were scored using nuclei from multiple experiments. (c) 3-D reconstituted nuclei (as in Fig. 1 a) are divided in two spaces of similar volume and the frequency of FISH signals is scored. ARS1412 (5, 5), ChrIV-908 (10, 5), and Y′ (22, 7). (d) The localization of six late-activated (dark columns) and six early-activated (light columns) origins has been quantified by distance-to-edge measurements as described in a. For each zone, the theoretical random signal distribution, based on surface area, has been subtracted from the corresponding signal frequency. A value of 0 corresponds to random distribution of signals; numbers > and < 0 indicate enrichment and depletion, respectively. Columns from left to right (brackets: n = signals, nuclei): ARS1412 (106, 76), ARS1413 (143, 94), ChrIV-210 (112, 78), ChrIV-257 (111, 77), ARS603 (64, 54), ChrX-305 (189, 123) ARS1 (43, 32), ChrIV-908 (83, 64), ChrIV-1153 (123, 86), ARS606 (83, 66), ARS607 (47, 37), and ChrX-613 (81, 63). A χ2 test demonstrates that four out of six late origins and none of the early origin FISH signals differ significantly from a random distribution to P < 0.05. When taken together, the localization of the pooled late origins in zone 1 is significantly nonrandom and is significantly different from the localization of the pooled early origins (P < 0.001).

Mentions: FISH signals were localized relative to the nuclear periphery by the use of concentric circles that either define five regions within the nucleus of fixed area or two zones of equal area. For the latter, an internal circle of radius 0.71 divides the surface area into two equal parts. A second method for scoring perinuclear localization was performed by image processing as follows. The antinuclear pore signal was computationally expanded to occupy a zone that contains 50% of the surface area of the nucleus, while leaving the FISH signals unmodified. FISH signals overlapping >50% with the enlarged pore zone were scored as peripheral. Finally, to analyze the position of the ARS501-excision cassette or GFP-tagged origins relative to either the telomere proximal locus on chromosome V or to the nuclear periphery, the line profile tool of the 510 Confocal software version 2.5 was used. If the minimal distance between the FISH signal and the nuclear pore was <0.29×, the nuclear radius, it was scored as peripheral. In three-dimensional reconstituted nuclei, the presence of signals within two equal volumes defining a peripheral and an internal sphere was quantified. FISH signals were scored on orthogonal representations of the nucleus. Centromere FISH signals were scored as clustered when found within a circle containing 16% of the nuclear surface area. Standard χ2 tests were performed to determine the statistical significance of differences between the FISH signal distribution in the nucleus and a random distribution. Following the convention to avoid frequencies less than five in any given group, frequencies in zones 4 and 5 (see Fig. 3 a) have been combined for the χ2 test.


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)

Late replication origins are enriched near the nuclear periphery. FISH signals were quantified on equatorial confocal sections of diploid strain GA-116, for ARS1412, ChrIV-908, and Y′-containing telomeres (Fig. 2b, Fig. c, and Fig. e) relative to the zones indicated in a–c. ARS1412 and the subtelomeric Y′ elements are late replicating, while ChrIV-908 is early. (a) Distance to edge is measured by the use of five zones, and data are represented in a bar graph as a percentage of signals per zone, each of which has a different area. The line corresponds to theoretical values for randomly distributed signals and is proportional to the surface area of each zone. From left to right (in brackets: n = signals, nuclei): ARS1412 (106, 76), ChrIV-908 (83, 64), and Y′ (264, 66). Each experiment has been performed at least twice and error bars represent standard deviations. Analysis using a standard χ2 test reveals a statistically significant nonrandom localization for ARS1412 (P < 0.05) and Y′ FISH probes (P < 0.001), but not for ChrIV-908 (P = 0.1). (b) The nuclei are divided into two zones of equal surface area: peripheral and internal. FISH signal localization was quantified on a computer graphic representation of 2-D confocal images in which the nuclear pore staining was enlarged to create a peripheral zone that comprises 50% of the original nuclear surface. FISH signals found within this zone were scored as peripheral. ARS1412 (43, 42), ChrIV-908 (59, 44), and Y′ (149, 44). ARS1412 and Y′ signals were scored using nuclei from multiple experiments. (c) 3-D reconstituted nuclei (as in Fig. 1 a) are divided in two spaces of similar volume and the frequency of FISH signals is scored. ARS1412 (5, 5), ChrIV-908 (10, 5), and Y′ (22, 7). (d) The localization of six late-activated (dark columns) and six early-activated (light columns) origins has been quantified by distance-to-edge measurements as described in a. For each zone, the theoretical random signal distribution, based on surface area, has been subtracted from the corresponding signal frequency. A value of 0 corresponds to random distribution of signals; numbers > and < 0 indicate enrichment and depletion, respectively. Columns from left to right (brackets: n = signals, nuclei): ARS1412 (106, 76), ARS1413 (143, 94), ChrIV-210 (112, 78), ChrIV-257 (111, 77), ARS603 (64, 54), ChrX-305 (189, 123) ARS1 (43, 32), ChrIV-908 (83, 64), ChrIV-1153 (123, 86), ARS606 (83, 66), ARS607 (47, 37), and ChrX-613 (81, 63). A χ2 test demonstrates that four out of six late origins and none of the early origin FISH signals differ significantly from a random distribution to P < 0.05. When taken together, the localization of the pooled late origins in zone 1 is significantly nonrandom and is significantly different from the localization of the pooled early origins (P < 0.001).
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Related In: Results  -  Collection

Show All Figures
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Figure 3: Late replication origins are enriched near the nuclear periphery. FISH signals were quantified on equatorial confocal sections of diploid strain GA-116, for ARS1412, ChrIV-908, and Y′-containing telomeres (Fig. 2b, Fig. c, and Fig. e) relative to the zones indicated in a–c. ARS1412 and the subtelomeric Y′ elements are late replicating, while ChrIV-908 is early. (a) Distance to edge is measured by the use of five zones, and data are represented in a bar graph as a percentage of signals per zone, each of which has a different area. The line corresponds to theoretical values for randomly distributed signals and is proportional to the surface area of each zone. From left to right (in brackets: n = signals, nuclei): ARS1412 (106, 76), ChrIV-908 (83, 64), and Y′ (264, 66). Each experiment has been performed at least twice and error bars represent standard deviations. Analysis using a standard χ2 test reveals a statistically significant nonrandom localization for ARS1412 (P < 0.05) and Y′ FISH probes (P < 0.001), but not for ChrIV-908 (P = 0.1). (b) The nuclei are divided into two zones of equal surface area: peripheral and internal. FISH signal localization was quantified on a computer graphic representation of 2-D confocal images in which the nuclear pore staining was enlarged to create a peripheral zone that comprises 50% of the original nuclear surface. FISH signals found within this zone were scored as peripheral. ARS1412 (43, 42), ChrIV-908 (59, 44), and Y′ (149, 44). ARS1412 and Y′ signals were scored using nuclei from multiple experiments. (c) 3-D reconstituted nuclei (as in Fig. 1 a) are divided in two spaces of similar volume and the frequency of FISH signals is scored. ARS1412 (5, 5), ChrIV-908 (10, 5), and Y′ (22, 7). (d) The localization of six late-activated (dark columns) and six early-activated (light columns) origins has been quantified by distance-to-edge measurements as described in a. For each zone, the theoretical random signal distribution, based on surface area, has been subtracted from the corresponding signal frequency. A value of 0 corresponds to random distribution of signals; numbers > and < 0 indicate enrichment and depletion, respectively. Columns from left to right (brackets: n = signals, nuclei): ARS1412 (106, 76), ARS1413 (143, 94), ChrIV-210 (112, 78), ChrIV-257 (111, 77), ARS603 (64, 54), ChrX-305 (189, 123) ARS1 (43, 32), ChrIV-908 (83, 64), ChrIV-1153 (123, 86), ARS606 (83, 66), ARS607 (47, 37), and ChrX-613 (81, 63). A χ2 test demonstrates that four out of six late origins and none of the early origin FISH signals differ significantly from a random distribution to P < 0.05. When taken together, the localization of the pooled late origins in zone 1 is significantly nonrandom and is significantly different from the localization of the pooled early origins (P < 0.001).
Mentions: FISH signals were localized relative to the nuclear periphery by the use of concentric circles that either define five regions within the nucleus of fixed area or two zones of equal area. For the latter, an internal circle of radius 0.71 divides the surface area into two equal parts. A second method for scoring perinuclear localization was performed by image processing as follows. The antinuclear pore signal was computationally expanded to occupy a zone that contains 50% of the surface area of the nucleus, while leaving the FISH signals unmodified. FISH signals overlapping >50% with the enlarged pore zone were scored as peripheral. Finally, to analyze the position of the ARS501-excision cassette or GFP-tagged origins relative to either the telomere proximal locus on chromosome V or to the nuclear periphery, the line profile tool of the 510 Confocal software version 2.5 was used. If the minimal distance between the FISH signal and the nuclear pore was <0.29×, the nuclear radius, it was scored as peripheral. In three-dimensional reconstituted nuclei, the presence of signals within two equal volumes defining a peripheral and an internal sphere was quantified. FISH signals were scored on orthogonal representations of the nucleus. Centromere FISH signals were scored as clustered when found within a circle containing 16% of the nuclear surface area. Standard χ2 tests were performed to determine the statistical significance of differences between the FISH signal distribution in the nucleus and a random distribution. Following the convention to avoid frequencies less than five in any given group, frequencies in zones 4 and 5 (see Fig. 3 a) have been combined for the χ2 test.

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