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DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts.

Labit H, Perewoska I, Germe T, Hyrien O, Marheineke K - Nucleic Acids Res. (2008)

Bottom Line: However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres.The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure.These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Biology Department, Laboratory of Molecular Genetics, CNRS UMR 8541, 46, rue d'Ulm, 75005 Paris, France.

ABSTRACT
Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

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Different replication patterns of sperm nuclei in Xenopus egg extracts. (A–F) Examples of representative foci patterns. Sperm nuclei were labelled early (A–D, at 16 min) or late (E and F, at 65 min) in S phase by incorporation of rhodamine–dUTP for 2 min (Z projections of 30 stacks at 0.2 μm interval), bar = 3 μm. (G) After pulse labelling of nuclei throughout S phase with rhodamine–dUTP, the different patterns of replication foci were scored (150 nuclei per time point; white: no foci, lines: few foci, grey: many punctuate foci, black: uniform staining).
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Figure 1: Different replication patterns of sperm nuclei in Xenopus egg extracts. (A–F) Examples of representative foci patterns. Sperm nuclei were labelled early (A–D, at 16 min) or late (E and F, at 65 min) in S phase by incorporation of rhodamine–dUTP for 2 min (Z projections of 30 stacks at 0.2 μm interval), bar = 3 μm. (G) After pulse labelling of nuclei throughout S phase with rhodamine–dUTP, the different patterns of replication foci were scored (150 nuclei per time point; white: no foci, lines: few foci, grey: many punctuate foci, black: uniform staining).

Mentions: Replication foci in sperm nuclei replicating in Xenopus egg extracts were visualized by high resolution fluorescence microscopy after short pulses of rhodamine–dUTP early and late in S phase. At the onset of S phase (16 min) we observed three different patterns of label incorporation due to the slight asynchronous entry into S phase: few punctuate foci (Figure 1A), many punctuate foci (Figure 1B and C) and bright nuclei whose individual foci were too numerous to be resolved (Figure 1D). Very late in S phase (65 min), discrete foci became again visible (Figure 1E and F). We also found variations in the size and distribution of foci: discrete foci polarized at one end of the nucleus (Figure 1B) were observed in ∼5% of total nuclei in both early and late S phase (their percentage varied between experiments). Some nuclei also presented few large foci scattered among a majority of smaller foci late in S phase (Figure 1F). To determine if the three patterns (few punctuate, many punctuate and bright) represented nuclei having progressed through S phase to different extents, their percentages were counted at different stages of S phase (Figure 1G). Nuclei with few or many individual foci predominated during very early S phase; they were quickly replaced by uniformly labelled nuclei by mid S phase and only reappeared late in S phase. This temporal succession of patterns suggests that under our experimental conditions, we are able to resolve the progressive activation and disappearance of replication foci during early and late S phase, respectively, and that foci are too numerous and/or too bright in mid S phase to be resolved.Figure 1.


DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts.

Labit H, Perewoska I, Germe T, Hyrien O, Marheineke K - Nucleic Acids Res. (2008)

Different replication patterns of sperm nuclei in Xenopus egg extracts. (A–F) Examples of representative foci patterns. Sperm nuclei were labelled early (A–D, at 16 min) or late (E and F, at 65 min) in S phase by incorporation of rhodamine–dUTP for 2 min (Z projections of 30 stacks at 0.2 μm interval), bar = 3 μm. (G) After pulse labelling of nuclei throughout S phase with rhodamine–dUTP, the different patterns of replication foci were scored (150 nuclei per time point; white: no foci, lines: few foci, grey: many punctuate foci, black: uniform staining).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Different replication patterns of sperm nuclei in Xenopus egg extracts. (A–F) Examples of representative foci patterns. Sperm nuclei were labelled early (A–D, at 16 min) or late (E and F, at 65 min) in S phase by incorporation of rhodamine–dUTP for 2 min (Z projections of 30 stacks at 0.2 μm interval), bar = 3 μm. (G) After pulse labelling of nuclei throughout S phase with rhodamine–dUTP, the different patterns of replication foci were scored (150 nuclei per time point; white: no foci, lines: few foci, grey: many punctuate foci, black: uniform staining).
Mentions: Replication foci in sperm nuclei replicating in Xenopus egg extracts were visualized by high resolution fluorescence microscopy after short pulses of rhodamine–dUTP early and late in S phase. At the onset of S phase (16 min) we observed three different patterns of label incorporation due to the slight asynchronous entry into S phase: few punctuate foci (Figure 1A), many punctuate foci (Figure 1B and C) and bright nuclei whose individual foci were too numerous to be resolved (Figure 1D). Very late in S phase (65 min), discrete foci became again visible (Figure 1E and F). We also found variations in the size and distribution of foci: discrete foci polarized at one end of the nucleus (Figure 1B) were observed in ∼5% of total nuclei in both early and late S phase (their percentage varied between experiments). Some nuclei also presented few large foci scattered among a majority of smaller foci late in S phase (Figure 1F). To determine if the three patterns (few punctuate, many punctuate and bright) represented nuclei having progressed through S phase to different extents, their percentages were counted at different stages of S phase (Figure 1G). Nuclei with few or many individual foci predominated during very early S phase; they were quickly replaced by uniformly labelled nuclei by mid S phase and only reappeared late in S phase. This temporal succession of patterns suggests that under our experimental conditions, we are able to resolve the progressive activation and disappearance of replication foci during early and late S phase, respectively, and that foci are too numerous and/or too bright in mid S phase to be resolved.Figure 1.

Bottom Line: However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres.The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure.These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

View Article: PubMed Central - PubMed

Affiliation: Ecole Normale Supérieure, Biology Department, Laboratory of Molecular Genetics, CNRS UMR 8541, 46, rue d'Ulm, 75005 Paris, France.

ABSTRACT
Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

Show MeSH