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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 replicating origins and telomeres do not colocalize at the periphery. Representative confocal images of the midsection of fixed yeast cells hybridized with FISH probes for the subtelomeric Y′ element and either (a) ARS1412, (b) the silent mating type locus HML, or (c) ChrIV-210. In d controls for maximal colocalization and stochastic coincidence are shown. For the latter, detection of a Y′ probe (red) was combined with the centromere of chromosome 8 (CEN8, green). For maximal colocalization values, cells were probed with identical Y′ DNAs labeled with dig-dUTP (green) or Alexa546-dUTP (red). The different combinations of probes were quantified for colocalization and results are shown in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
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Figure 5: Late replicating origins and telomeres do not colocalize at the periphery. Representative confocal images of the midsection of fixed yeast cells hybridized with FISH probes for the subtelomeric Y′ element and either (a) ARS1412, (b) the silent mating type locus HML, or (c) ChrIV-210. In d controls for maximal colocalization and stochastic coincidence are shown. For the latter, detection of a Y′ probe (red) was combined with the centromere of chromosome 8 (CEN8, green). For maximal colocalization values, cells were probed with identical Y′ DNAs labeled with dig-dUTP (green) or Alexa546-dUTP (red). The different combinations of probes were quantified for colocalization and results are shown in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.

Mentions: Since both internal late-activated origins and telomeres are enriched at the nuclear periphery in G1, we asked whether they localize to common sites by using codetection of differentially labeled FISH probes. As a control for multiple signals that we do not expect to colocalize, we first quantified the frequency with which two probes that recognize the centromere on chromosome VIII (CEN8) and the Y′ subtelomeric region overlap by ≥25% (Fig. 5 d, Table ). Using this criterion, 27% of the centromere signals were found to coincide with Y′ signals. In contrast, 88% colocalization was obtained using two differently labeled Y′ probes. This latter value indicates the experimental maximum for identical signals, while 27% most likely reflects the stochastic coincidence. When nontelomeric, late firing origins were tested in combination with the same Y′ probe, we found that only 33% of the signals coincide, a background equivalent to that obtained with CEN8. Similar frequencies of overlap were calculated for the late firing origins in combination with the Y′ probe (see probes ARS603, 1412, 1413, and ChrIV-257; Fig. 5, a and c, and Table ), indicating that in general, nontelomeric, late origins are not associated with telomeres, despite the fact that both are enriched in a perinuclear zone. Recent findings suggest that telomeres associate with the nuclear envelope through yKu (Laroche et al. 1998) and the large coiled-coil proteins Mlp1/2 (Galy et al. 2000). The lack of coincidence of Y′ elements and late origins, suggests that the latter associate with the nuclear periphery in a manner distinct from that which tethers telomeres.


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 replicating origins and telomeres do not colocalize at the periphery. Representative confocal images of the midsection of fixed yeast cells hybridized with FISH probes for the subtelomeric Y′ element and either (a) ARS1412, (b) the silent mating type locus HML, or (c) ChrIV-210. In d controls for maximal colocalization and stochastic coincidence are shown. For the latter, detection of a Y′ probe (red) was combined with the centromere of chromosome 8 (CEN8, green). For maximal colocalization values, cells were probed with identical Y′ DNAs labeled with dig-dUTP (green) or Alexa546-dUTP (red). The different combinations of probes were quantified for colocalization and results are shown in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
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Related In: Results  -  Collection

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Figure 5: Late replicating origins and telomeres do not colocalize at the periphery. Representative confocal images of the midsection of fixed yeast cells hybridized with FISH probes for the subtelomeric Y′ element and either (a) ARS1412, (b) the silent mating type locus HML, or (c) ChrIV-210. In d controls for maximal colocalization and stochastic coincidence are shown. For the latter, detection of a Y′ probe (red) was combined with the centromere of chromosome 8 (CEN8, green). For maximal colocalization values, cells were probed with identical Y′ DNAs labeled with dig-dUTP (green) or Alexa546-dUTP (red). The different combinations of probes were quantified for colocalization and results are shown in Table . Images were collected on an LSM 410 confocal microscope. Scale bar: 2 μm.
Mentions: Since both internal late-activated origins and telomeres are enriched at the nuclear periphery in G1, we asked whether they localize to common sites by using codetection of differentially labeled FISH probes. As a control for multiple signals that we do not expect to colocalize, we first quantified the frequency with which two probes that recognize the centromere on chromosome VIII (CEN8) and the Y′ subtelomeric region overlap by ≥25% (Fig. 5 d, Table ). Using this criterion, 27% of the centromere signals were found to coincide with Y′ signals. In contrast, 88% colocalization was obtained using two differently labeled Y′ probes. This latter value indicates the experimental maximum for identical signals, while 27% most likely reflects the stochastic coincidence. When nontelomeric, late firing origins were tested in combination with the same Y′ probe, we found that only 33% of the signals coincide, a background equivalent to that obtained with CEN8. Similar frequencies of overlap were calculated for the late firing origins in combination with the Y′ probe (see probes ARS603, 1412, 1413, and ChrIV-257; Fig. 5, a and c, and Table ), indicating that in general, nontelomeric, late origins are not associated with telomeres, despite the fact that both are enriched in a perinuclear zone. Recent findings suggest that telomeres associate with the nuclear envelope through yKu (Laroche et al. 1998) and the large coiled-coil proteins Mlp1/2 (Galy et al. 2000). The lack of coincidence of Y′ elements and late origins, suggests that the latter associate with the nuclear periphery in a manner distinct from that which tethers telomeres.

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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