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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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Colocalization of replication foci in two successive S phases in cycling extracts. (A) Labelling scheme: sperm nuclei (100 nuclei/µl) were incubated in egg extracts low speed supernatant (LSS) in the absence of cycloheximide, pulse labelled early in the first S phase with rhodamine-dUTP (16–18 min, red) and early in the second S phase with biotin-dATP (65–80 min, green). Replication foci in two different nuclei labelled in the first S phase (B and C), second S phase (D and E), merged images of first and second S phases (F and G), Hoechst (H and I), bar = 2 µm. (J) Quantitative colocalization analysis: the fraction of colocalized foci was calculated from 50 nuclei. Red curve shows a fitted Gaussian distribution.
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Figure 3: Colocalization of replication foci in two successive S phases in cycling extracts. (A) Labelling scheme: sperm nuclei (100 nuclei/µl) were incubated in egg extracts low speed supernatant (LSS) in the absence of cycloheximide, pulse labelled early in the first S phase with rhodamine-dUTP (16–18 min, red) and early in the second S phase with biotin-dATP (65–80 min, green). Replication foci in two different nuclei labelled in the first S phase (B and C), second S phase (D and E), merged images of first and second S phases (F and G), Hoechst (H and I), bar = 2 µm. (J) Quantitative colocalization analysis: the fraction of colocalized foci was calculated from 50 nuclei. Red curve shows a fitted Gaussian distribution.

Mentions: Having shown that replication foci are activated in a progressive manner during early S phase, we examined if this order was reproducible between two successive S phases in cycling egg extracts, which are able to perform several rounds of alternating S and M phases (26). Sperm nuclei were pulse labelled at the beginning of the first S phase with digoxigenin–dUTP, chased with dTTP and pulsed labelled again with biotin–dATP at the beginning of the second S phase (Figure 3A). The kinetics of entry into the intervening mitosis was simultaneously monitored by direct fluorescence microscopy to control that the second label was added at the appropriate time. No incorporation of biotin–dATP was observed when entry into mitosis was prevented using cycloheximide (data not shown). However, in cycling extracts we observed a high degree of colocalization of the two labels within one nucleus, showing that the first active foci at the beginning of one S phase were also the first to be activated at the beginning of the next (Figure 3B–I). In order to quantify the colocalization of replication foci between two S phases we analysed 50 nuclei showing early S phase replication pattern. The distribution of colocalization values fitted a Gaussian distribution and a mean of 92% (± 6.9%) colocalization was calculated (Figure 3J). This experiment shows that the temporal order of foci appearance is maintained from one cell cycle to the other. We conclude that foci are activated in a non-random, thus deterministic order in Xenopus egg extracts.Figure 3.


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)

Colocalization of replication foci in two successive S phases in cycling extracts. (A) Labelling scheme: sperm nuclei (100 nuclei/µl) were incubated in egg extracts low speed supernatant (LSS) in the absence of cycloheximide, pulse labelled early in the first S phase with rhodamine-dUTP (16–18 min, red) and early in the second S phase with biotin-dATP (65–80 min, green). Replication foci in two different nuclei labelled in the first S phase (B and C), second S phase (D and E), merged images of first and second S phases (F and G), Hoechst (H and I), bar = 2 µm. (J) Quantitative colocalization analysis: the fraction of colocalized foci was calculated from 50 nuclei. Red curve shows a fitted Gaussian distribution.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: Colocalization of replication foci in two successive S phases in cycling extracts. (A) Labelling scheme: sperm nuclei (100 nuclei/µl) were incubated in egg extracts low speed supernatant (LSS) in the absence of cycloheximide, pulse labelled early in the first S phase with rhodamine-dUTP (16–18 min, red) and early in the second S phase with biotin-dATP (65–80 min, green). Replication foci in two different nuclei labelled in the first S phase (B and C), second S phase (D and E), merged images of first and second S phases (F and G), Hoechst (H and I), bar = 2 µm. (J) Quantitative colocalization analysis: the fraction of colocalized foci was calculated from 50 nuclei. Red curve shows a fitted Gaussian distribution.
Mentions: Having shown that replication foci are activated in a progressive manner during early S phase, we examined if this order was reproducible between two successive S phases in cycling egg extracts, which are able to perform several rounds of alternating S and M phases (26). Sperm nuclei were pulse labelled at the beginning of the first S phase with digoxigenin–dUTP, chased with dTTP and pulsed labelled again with biotin–dATP at the beginning of the second S phase (Figure 3A). The kinetics of entry into the intervening mitosis was simultaneously monitored by direct fluorescence microscopy to control that the second label was added at the appropriate time. No incorporation of biotin–dATP was observed when entry into mitosis was prevented using cycloheximide (data not shown). However, in cycling extracts we observed a high degree of colocalization of the two labels within one nucleus, showing that the first active foci at the beginning of one S phase were also the first to be activated at the beginning of the next (Figure 3B–I). In order to quantify the colocalization of replication foci between two S phases we analysed 50 nuclei showing early S phase replication pattern. The distribution of colocalization values fitted a Gaussian distribution and a mean of 92% (± 6.9%) colocalization was calculated (Figure 3J). This experiment shows that the temporal order of foci appearance is maintained from one cell cycle to the other. We conclude that foci are activated in a non-random, thus deterministic order in Xenopus egg extracts.Figure 3.

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
Related in: MedlinePlus