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
The rDNA replicates later than bulk genomic DNA and has a different chromatin structure. (A) Merged images of representative rDNA repeats in mid S phase. Green, rDNA probe; red: replication bubbles labelled by biotin-dUTP incorporation from 0 to 35 min and chased with dTTP (mid S phase), bar = 3 kb. (B) Replication extent of rDNA repeats and whole genomic DNA as analysed by combing. (C) Micrococcal digestion of sperm nuclei assembled in egg extracts in the presence of geminin. DNA was digested for the indicated time points, electrophoresed and hybridized either with a genomic DNA probe (left) or a full-length rDNA repeat probe (right). Positions of mono- and oligomeric nucleosomal DNA fragments are indicated on the left.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: The rDNA replicates later than bulk genomic DNA and has a different chromatin structure. (A) Merged images of representative rDNA repeats in mid S phase. Green, rDNA probe; red: replication bubbles labelled by biotin-dUTP incorporation from 0 to 35 min and chased with dTTP (mid S phase), bar = 3 kb. (B) Replication extent of rDNA repeats and whole genomic DNA as analysed by combing. (C) Micrococcal digestion of sperm nuclei assembled in egg extracts in the presence of geminin. DNA was digested for the indicated time points, electrophoresed and hybridized either with a genomic DNA probe (left) or a full-length rDNA repeat probe (right). Positions of mono- and oligomeric nucleosomal DNA fragments are indicated on the left.

Mentions: In order to determine the replication timing of the rDNA sequences relative to whole genome sequences, we compared the mean replication extent of the rDNA with that of the whole genome by summing the length of the bubbles and dividing by the total length of the respective DNA molecules. For this experiment, sperm nuclei were labelled with biotin–dUTP, stopped early and midway through S phase. Biotin-labelled replication bubbles were visualized in red. The rDNA (Figure 6A) was visualized by hybridization with a B fragment probe labelled by digoxigenin (in green). The whole genomic DNA was visualized using an anti-guanosine antibody (not shown). The replication extent of the rDNA was significantly lower than the replication extent of the whole genome in both early and mid S phase (Figure 6B: early S, 6.2% versus 11.5%, P < 0.05; mid S, 53.2% versus 64.4%, P < 0.01). Although the ETED (mean 7.5 kb versus 8 kb) and eye sizes (1.1 kb versus 1.2 kb) in early S phase were similar, the overall density of forks was lower in the rDNA than in the whole genome (9.5 versus 15.9 forks/100 kb) in early S phase. This suggests that origins in the rDNA are activated in clusters, as in the bulk of the genome, but at a slower pace.Figure 6.


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 rDNA replicates later than bulk genomic DNA and has a different chromatin structure. (A) Merged images of representative rDNA repeats in mid S phase. Green, rDNA probe; red: replication bubbles labelled by biotin-dUTP incorporation from 0 to 35 min and chased with dTTP (mid S phase), bar = 3 kb. (B) Replication extent of rDNA repeats and whole genomic DNA as analysed by combing. (C) Micrococcal digestion of sperm nuclei assembled in egg extracts in the presence of geminin. DNA was digested for the indicated time points, electrophoresed and hybridized either with a genomic DNA probe (left) or a full-length rDNA repeat probe (right). Positions of mono- and oligomeric nucleosomal DNA fragments are indicated on the left.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: The rDNA replicates later than bulk genomic DNA and has a different chromatin structure. (A) Merged images of representative rDNA repeats in mid S phase. Green, rDNA probe; red: replication bubbles labelled by biotin-dUTP incorporation from 0 to 35 min and chased with dTTP (mid S phase), bar = 3 kb. (B) Replication extent of rDNA repeats and whole genomic DNA as analysed by combing. (C) Micrococcal digestion of sperm nuclei assembled in egg extracts in the presence of geminin. DNA was digested for the indicated time points, electrophoresed and hybridized either with a genomic DNA probe (left) or a full-length rDNA repeat probe (right). Positions of mono- and oligomeric nucleosomal DNA fragments are indicated on the left.
Mentions: In order to determine the replication timing of the rDNA sequences relative to whole genome sequences, we compared the mean replication extent of the rDNA with that of the whole genome by summing the length of the bubbles and dividing by the total length of the respective DNA molecules. For this experiment, sperm nuclei were labelled with biotin–dUTP, stopped early and midway through S phase. Biotin-labelled replication bubbles were visualized in red. The rDNA (Figure 6A) was visualized by hybridization with a B fragment probe labelled by digoxigenin (in green). The whole genomic DNA was visualized using an anti-guanosine antibody (not shown). The replication extent of the rDNA was significantly lower than the replication extent of the whole genome in both early and mid S phase (Figure 6B: early S, 6.2% versus 11.5%, P < 0.05; mid S, 53.2% versus 64.4%, P < 0.01). Although the ETED (mean 7.5 kb versus 8 kb) and eye sizes (1.1 kb versus 1.2 kb) in early S phase were similar, the overall density of forks was lower in the rDNA than in the whole genome (9.5 versus 15.9 forks/100 kb) in early S phase. This suggests that origins in the rDNA are activated in clusters, as in the bulk of the genome, but at a slower pace.Figure 6.

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