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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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The number of replication foci changes during S phase in Xenopus egg extracts. (A) Example nuclei labelled early with rhodamine–dUTP before (Z projection of 30 stacks, left) and after (right) deconvolution, bar = 1 μm. (B) Early and late replication foci in 50 nuclei were counted on deconvoluted images. Results are shown as box-and-whisker plots with vertical lines indicating the 98% range, boxes the 25–75th percentile and black horizontal lines the mean. (C) Box-and-whisker plots of the number of early (t = 16 min) replication foci in control and caffeine (5 mM) treated samples.
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Figure 2: The number of replication foci changes during S phase in Xenopus egg extracts. (A) Example nuclei labelled early with rhodamine–dUTP before (Z projection of 30 stacks, left) and after (right) deconvolution, bar = 1 μm. (B) Early and late replication foci in 50 nuclei were counted on deconvoluted images. Results are shown as box-and-whisker plots with vertical lines indicating the 98% range, boxes the 25–75th percentile and black horizontal lines the mean. (C) Box-and-whisker plots of the number of early (t = 16 min) replication foci in control and caffeine (5 mM) treated samples.

Mentions: To obtain more precise quantifications, replication foci were counted next on deconvoluted images after Z-stack projections (Figure 2A). Nuclei were pulse labelled with rhodamine–dUTP at two early (17 and 20 min) and two late (65 and 70 min) time points in S phase. The number of foci per nucleus increased from a few up to maximal 250 foci per nucleus early in S phase and decreased again late in S phase (Figure 2B). We next asked whether the ATM/ATR inhibitor caffeine affected the activation of replication foci in very early S phase. No significant effect of caffeine on the number of foci was observed (P = 0.35; Mann–Whitney test, Figure 2C). However, the mean pixel intensities of nuclei were higher in the presence of caffeine (data not shown), in agreement with the previously reported effect of caffeine on the rate of DNA synthesis (20,25).Figure 2.


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)

The number of replication foci changes during S phase in Xenopus egg extracts. (A) Example nuclei labelled early with rhodamine–dUTP before (Z projection of 30 stacks, left) and after (right) deconvolution, bar = 1 μm. (B) Early and late replication foci in 50 nuclei were counted on deconvoluted images. Results are shown as box-and-whisker plots with vertical lines indicating the 98% range, boxes the 25–75th percentile and black horizontal lines the mean. (C) Box-and-whisker plots of the number of early (t = 16 min) replication foci in control and caffeine (5 mM) treated samples.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553594&req=5

Figure 2: The number of replication foci changes during S phase in Xenopus egg extracts. (A) Example nuclei labelled early with rhodamine–dUTP before (Z projection of 30 stacks, left) and after (right) deconvolution, bar = 1 μm. (B) Early and late replication foci in 50 nuclei were counted on deconvoluted images. Results are shown as box-and-whisker plots with vertical lines indicating the 98% range, boxes the 25–75th percentile and black horizontal lines the mean. (C) Box-and-whisker plots of the number of early (t = 16 min) replication foci in control and caffeine (5 mM) treated samples.
Mentions: To obtain more precise quantifications, replication foci were counted next on deconvoluted images after Z-stack projections (Figure 2A). Nuclei were pulse labelled with rhodamine–dUTP at two early (17 and 20 min) and two late (65 and 70 min) time points in S phase. The number of foci per nucleus increased from a few up to maximal 250 foci per nucleus early in S phase and decreased again late in S phase (Figure 2B). We next asked whether the ATM/ATR inhibitor caffeine affected the activation of replication foci in very early S phase. No significant effect of caffeine on the number of foci was observed (P = 0.35; Mann–Whitney test, Figure 2C). However, the mean pixel intensities of nuclei were higher in the presence of caffeine (data not shown), in agreement with the previously reported effect of caffeine on the rate of DNA synthesis (20,25).Figure 2.

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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