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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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Preferential initiation in the transcription unit of the rDNA repeat. (A) Map of the X. laevis rDNA repeat and localization of probe B and A (grey boxes), GC plot with 55 bp sliding window; IGS, intergenic spacer. (B and D) Sperm nuclei were labelled for 30 min with digoxigenin–dUTP to mark replication eyes (in green) and combed DNA fibres were hybridized with biotinylated probe B (B) or probe A (D) (red); bar = 3 kb. (C) Origin counts on probe B and on the rest of the repeat (fragment A) and on probe A and the rest of the repeat (fragment B). A total of 5 Mb of rDNA was analysed.
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Figure 5: Preferential initiation in the transcription unit of the rDNA repeat. (A) Map of the X. laevis rDNA repeat and localization of probe B and A (grey boxes), GC plot with 55 bp sliding window; IGS, intergenic spacer. (B and D) Sperm nuclei were labelled for 30 min with digoxigenin–dUTP to mark replication eyes (in green) and combed DNA fibres were hybridized with biotinylated probe B (B) or probe A (D) (red); bar = 3 kb. (C) Origin counts on probe B and on the rest of the repeat (fragment A) and on probe A and the rest of the repeat (fragment B). A total of 5 Mb of rDNA was analysed.

Mentions: So far the replication programme was analysed on the whole genome level without distinguishing specific sequences. We therefore intended to study the timing programme at the rDNA repeat region, spanning 4 Mb on chromosome XII in Xenopus laevis. Replication of the rDNA locus in pre-MBT Xenopus embryos has been studied by 2D gel electrophoresis (12,16). Replication bubbles were detected everywhere along the rDNA repeat. Here, we re-investigated the distribution of initiation events along the rDNA repeat on single DNA fibres by DNA combing. Sperm chromatin replicating in egg extracts was labelled with digoxigenin–dUTP (t = 0–30 min), the DNA was purified and combed and the digoxigenin–labelled replication bubbles were detected with green fluorescent antibodies. The combed DNA was hybridized with a biotin-labelled fragment comprising the 3′ two-thirds of the rDNA transcription unit (probe B, Figure 5A) to reveal the rDNA repeats in red (Figure 5B). Initiation events were observed both within and outside fragment B, with a mean spacing of 9.4 kb, as expected. However, the frequency of replication initiation normalized to DNA length was 2.5-fold higher in fragment B than in the rest of the repeat (17.2 versus 7.1 events per 100 kb; P < 0.001; Figure 5C). As hybridization might have interfered with the detection of replication signals we also used a probe against the intergenic spacer of the transcription unit (Figure 5D) and analysed the distribution of initiation events on DNA fibres labelled with digoxigenin–dUTP (Figure 5C). Consistently, we found twice less initiations in the spacer region than in the gene region (6.3 versus 12.9 events per 100 kb; P < 0.001; Figure 5C). Similar results were obtained in a second independent experiment using the same two probes and in an experiment using a third probe against the 5′-end of transcription unit (data not shown). The intergenic spacer sequence is known to be very GC-rich (27). Since it has been reported that Xenopus origin recognition complex (ORC) preferentially binds to AT-rich sequences (28), we compared the GC/AT content of the intergenic spacer with the coding region. The sequences of the two regions have a different GC/AT content (IGS: 78%; transcription unit: 67.3% GC; Figure 5A). Overall, these results demonstrate a higher frequency of initiation in the transcription unit than in the intergenic spacer. We conclude that initiation in the rDNA repeat unit is not completely random before MBT in the absence of transcription.Figure 5.


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)

Preferential initiation in the transcription unit of the rDNA repeat. (A) Map of the X. laevis rDNA repeat and localization of probe B and A (grey boxes), GC plot with 55 bp sliding window; IGS, intergenic spacer. (B and D) Sperm nuclei were labelled for 30 min with digoxigenin–dUTP to mark replication eyes (in green) and combed DNA fibres were hybridized with biotinylated probe B (B) or probe A (D) (red); bar = 3 kb. (C) Origin counts on probe B and on the rest of the repeat (fragment A) and on probe A and the rest of the repeat (fragment B). A total of 5 Mb of rDNA was analysed.
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Figure 5: Preferential initiation in the transcription unit of the rDNA repeat. (A) Map of the X. laevis rDNA repeat and localization of probe B and A (grey boxes), GC plot with 55 bp sliding window; IGS, intergenic spacer. (B and D) Sperm nuclei were labelled for 30 min with digoxigenin–dUTP to mark replication eyes (in green) and combed DNA fibres were hybridized with biotinylated probe B (B) or probe A (D) (red); bar = 3 kb. (C) Origin counts on probe B and on the rest of the repeat (fragment A) and on probe A and the rest of the repeat (fragment B). A total of 5 Mb of rDNA was analysed.
Mentions: So far the replication programme was analysed on the whole genome level without distinguishing specific sequences. We therefore intended to study the timing programme at the rDNA repeat region, spanning 4 Mb on chromosome XII in Xenopus laevis. Replication of the rDNA locus in pre-MBT Xenopus embryos has been studied by 2D gel electrophoresis (12,16). Replication bubbles were detected everywhere along the rDNA repeat. Here, we re-investigated the distribution of initiation events along the rDNA repeat on single DNA fibres by DNA combing. Sperm chromatin replicating in egg extracts was labelled with digoxigenin–dUTP (t = 0–30 min), the DNA was purified and combed and the digoxigenin–labelled replication bubbles were detected with green fluorescent antibodies. The combed DNA was hybridized with a biotin-labelled fragment comprising the 3′ two-thirds of the rDNA transcription unit (probe B, Figure 5A) to reveal the rDNA repeats in red (Figure 5B). Initiation events were observed both within and outside fragment B, with a mean spacing of 9.4 kb, as expected. However, the frequency of replication initiation normalized to DNA length was 2.5-fold higher in fragment B than in the rest of the repeat (17.2 versus 7.1 events per 100 kb; P < 0.001; Figure 5C). As hybridization might have interfered with the detection of replication signals we also used a probe against the intergenic spacer of the transcription unit (Figure 5D) and analysed the distribution of initiation events on DNA fibres labelled with digoxigenin–dUTP (Figure 5C). Consistently, we found twice less initiations in the spacer region than in the gene region (6.3 versus 12.9 events per 100 kb; P < 0.001; Figure 5C). Similar results were obtained in a second independent experiment using the same two probes and in an experiment using a third probe against the 5′-end of transcription unit (data not shown). The intergenic spacer sequence is known to be very GC-rich (27). Since it has been reported that Xenopus origin recognition complex (ORC) preferentially binds to AT-rich sequences (28), we compared the GC/AT content of the intergenic spacer with the coding region. The sequences of the two regions have a different GC/AT content (IGS: 78%; transcription unit: 67.3% GC; Figure 5A). Overall, these results demonstrate a higher frequency of initiation in the transcription unit than in the intergenic spacer. We conclude that initiation in the rDNA repeat unit is not completely random before MBT in the absence of transcription.Figure 5.

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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