Labit H et al. (2008), DNA replication timing is deterministic at the ...

XB-ART-38330

Nucleic Acids Res 2008 Oct 01;3617:5623-34. doi: 10.1093/nar/gkn533.

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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 .

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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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Species referenced: Xenopus laevis
Genes referenced: antxr1 atm atr gmnn lss

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	Figure 1. Different replication patterns of sperm nuclei in Xenopus egg extracts. (AâF) Examples of representative foci patterns. Sperm nuclei were labelled early (AâD, at 16 min) or late (E and F, at 65 min) in S phase by incorporation of rhodamineâdUTP for 2 min (Z projections of 30 stacks at 0.2 Î¼m interval), bar = 3 Î¼m. (G) After pulse labelling of nuclei throughout S phase with rhodamineâdUTP, the different patterns of replication foci were scored (150 nuclei per time point; white: no foci, lines: few foci, grey: many punctuate foci, black: uniform staining).
	Figure 2. The number of replication foci changes during S phase in Xenopus egg extracts. (A) Example nuclei labelled early with rhodamineâdUTP before (Z projection of 30 stacks, left) and after (right) deconvolution, bar = 1 Î¼m. (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 25â75th 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.
	Figure 3. Colocalization of replication foci in two successive S phases in cycling extracts. (A) Labelling scheme: sperm nuclei (100 nuclei/Âµl) 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 (16â18 min, red) and early in the second S phase with biotin-dATP (65â80 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 Âµm. (J) Quantitative colocalization analysis: the fraction of colocalized foci was calculated from 50 nuclei. Red curve shows a fitted Gaussian distribution.
	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/Âµl in cycling egg extracts in the presence of [Î±-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).
	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 digoxigeninâdUTP 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.
	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.
	Figure 7. Model of replication timing in the Xenopus in vitro system. Replication timing is maintained during subsequent S phases at the level of replication foci and large chromatin domains, but not at the level of replication origins.

References [+] :

Anglana, Dynamics of DNA replication in mammalian somatic cells: nucleotide pool modulates origin choice and interorigin spacing. 2003, Pubmed