Limits...
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).

Show MeSH

Related in: MedlinePlus

Activation of origins in two successive S phases. (A) Labelling scheme for colocalization of origins used in two consecutive S phases by DNA combing. (B) Replication kinetics: sperm nuclei were incubated at 100 nuclei/µl in cycling egg extracts in the presence of [α-32P]dATP and DNA synthesis was quantified. The time at which label and chase nucleotides were added is indicated by arrows. (C) Representative fibres labelled early in two consecutive cell cycles (green = digoxigenin-dUTP and red = biotin-dATP), as detailed in (A). The faint, continuous green line in the first panel corresponds to whole DNA stained with YOYO-1, the panel just below represents the same fibre (different contrast enhancement). Three different types of ETED (ETEDdig, ETEDbio, ETEDdigbio) were measured, as indicated above the images; bar = 10 kb. (D) Distributions of ETED between origins activated in the first S phase only (ETEDdig, green circles), second S phase only (ETEDbio, red squares) and between origins activated in the two consecutive S phases (all ETED or ETEDdigbio, blue diamonds).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2553594&req=5

Figure 4: Activation of origins in two successive S phases. (A) Labelling scheme for colocalization of origins used in two consecutive S phases by DNA combing. (B) Replication kinetics: sperm nuclei were incubated at 100 nuclei/µl in cycling egg extracts in the presence of [α-32P]dATP and DNA synthesis was quantified. The time at which label and chase nucleotides were added is indicated by arrows. (C) Representative fibres labelled early in two consecutive cell cycles (green = digoxigenin-dUTP and red = biotin-dATP), as detailed in (A). The faint, continuous green line in the first panel corresponds to whole DNA stained with YOYO-1, the panel just below represents the same fibre (different contrast enhancement). Three different types of ETED (ETEDdig, ETEDbio, ETEDdigbio) were measured, as indicated above the images; bar = 10 kb. (D) Distributions of ETED between origins activated in the first S phase only (ETEDdig, green circles), second S phase only (ETEDbio, red squares) and between origins activated in the two consecutive S phases (all ETED or ETEDdigbio, blue diamonds).

Mentions: We next investigated whether the colocalization of labels incorporated at the beginning of two consecutive S phases, as observed at the level of replication foci, could also be observed at the level of origins and origin clusters on combed DNA fibres. Early firing replication origins in the first S phase were labelled by a pulse of digoxigenin–dUTP, followed by a chase with dTTP (t = 0–24 min), and early firing origins in the second S phase were labelled by a pulse of biotin–dATP, followed by a chase with dATP (t = 60–90 min) (Figure 4A). Several control experiments were performed in parallel. First, the kinetics of entry into mitosis was simultaneously monitored by fluorescence microscopy of whole nuclei in the presence of Hoechst. Second, replication kinetics was monitored by [α-32P]dATP incorporation (Figure 4B) and by DNA combing in both rounds (not shown). Both S phases were found to be efficient (>50%). The clear plateau of incorporation between 45 and 75 min shows that biotin–dATP was neither added too early nor too late in order to label origins in the second S phase. Third, in order to exclude that some nuclei had not finished replication at the time of biotin–ATP addition, its incorporation was assessed in the presence of cycloheximide, which prevents exit from the first interphase. We found that biotin–dATP incorporation on single fibres was negligible. Finally, incorporation of digoxigenin–dUTP in the first S phase had no effect on the cycling kinetics and the incorporation of biotin–dATP during the second S phase (data not shown).Figure 4.


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)

Activation of origins in two successive S phases. (A) Labelling scheme for colocalization of origins used in two consecutive S phases by DNA combing. (B) Replication kinetics: sperm nuclei were incubated at 100 nuclei/µl in cycling egg extracts in the presence of [α-32P]dATP and DNA synthesis was quantified. The time at which label and chase nucleotides were added is indicated by arrows. (C) Representative fibres labelled early in two consecutive cell cycles (green = digoxigenin-dUTP and red = biotin-dATP), as detailed in (A). The faint, continuous green line in the first panel corresponds to whole DNA stained with YOYO-1, the panel just below represents the same fibre (different contrast enhancement). Three different types of ETED (ETEDdig, ETEDbio, ETEDdigbio) were measured, as indicated above the images; bar = 10 kb. (D) Distributions of ETED between origins activated in the first S phase only (ETEDdig, green circles), second S phase only (ETEDbio, red squares) and between origins activated in the two consecutive S phases (all ETED or ETEDdigbio, blue diamonds).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Activation of origins in two successive S phases. (A) Labelling scheme for colocalization of origins used in two consecutive S phases by DNA combing. (B) Replication kinetics: sperm nuclei were incubated at 100 nuclei/µl in cycling egg extracts in the presence of [α-32P]dATP and DNA synthesis was quantified. The time at which label and chase nucleotides were added is indicated by arrows. (C) Representative fibres labelled early in two consecutive cell cycles (green = digoxigenin-dUTP and red = biotin-dATP), as detailed in (A). The faint, continuous green line in the first panel corresponds to whole DNA stained with YOYO-1, the panel just below represents the same fibre (different contrast enhancement). Three different types of ETED (ETEDdig, ETEDbio, ETEDdigbio) were measured, as indicated above the images; bar = 10 kb. (D) Distributions of ETED between origins activated in the first S phase only (ETEDdig, green circles), second S phase only (ETEDbio, red squares) and between origins activated in the two consecutive S phases (all ETED or ETEDdigbio, blue diamonds).
Mentions: We next investigated whether the colocalization of labels incorporated at the beginning of two consecutive S phases, as observed at the level of replication foci, could also be observed at the level of origins and origin clusters on combed DNA fibres. Early firing replication origins in the first S phase were labelled by a pulse of digoxigenin–dUTP, followed by a chase with dTTP (t = 0–24 min), and early firing origins in the second S phase were labelled by a pulse of biotin–dATP, followed by a chase with dATP (t = 60–90 min) (Figure 4A). Several control experiments were performed in parallel. First, the kinetics of entry into mitosis was simultaneously monitored by fluorescence microscopy of whole nuclei in the presence of Hoechst. Second, replication kinetics was monitored by [α-32P]dATP incorporation (Figure 4B) and by DNA combing in both rounds (not shown). Both S phases were found to be efficient (>50%). The clear plateau of incorporation between 45 and 75 min shows that biotin–dATP was neither added too early nor too late in order to label origins in the second S phase. Third, in order to exclude that some nuclei had not finished replication at the time of biotin–ATP addition, its incorporation was assessed in the presence of cycloheximide, which prevents exit from the first interphase. We found that biotin–dATP incorporation on single fibres was negligible. Finally, incorporation of digoxigenin–dUTP in the first S phase had no effect on the cycling kinetics and the incorporation of biotin–dATP during the second S phase (data not shown).Figure 4.

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