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Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels.

Thomson AM, Gillespie PJ, Blow JJ - J. Cell Biol. (2010)

Bottom Line: We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei.Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it.This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase.

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Replication foci number depends on Cdk levels. CHOC-400 nuclei were incubated in X. laevis egg extracts incubated at 10,000 nuclei/µl in egg extracts supplemented with the indicated concentrations of roscovitine (rosc) or cyclin A (cyc A). Parallel incubations were supplemented with α-[32P]dATP to measure total DNA synthesis. At 50 or 90 min, extract was pulsed for 5 min with Cy3-dUTP. (a–c) Representative images from the 50-min time point are shown. (d and e) The number and mean intensity of Cy3-labeled foci at 50 (d) and 90 (e) min were measured. Error bars indicate SEM between different nuclei.
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fig7: Replication foci number depends on Cdk levels. CHOC-400 nuclei were incubated in X. laevis egg extracts incubated at 10,000 nuclei/µl in egg extracts supplemented with the indicated concentrations of roscovitine (rosc) or cyclin A (cyc A). Parallel incubations were supplemented with α-[32P]dATP to measure total DNA synthesis. At 50 or 90 min, extract was pulsed for 5 min with Cy3-dUTP. (a–c) Representative images from the 50-min time point are shown. (d and e) The number and mean intensity of Cy3-labeled foci at 50 (d) and 90 (e) min were measured. Error bars indicate SEM between different nuclei.

Mentions: When progression into a new stage of the timing program occurs, new initiation events must occur in newly activated replication factories. Therefore, we investigated which of these aspects were most strongly affected when Cdk activity was varied. CHO nuclei replicating in vitro plus or minus roscovitine or cyclin A were pulsed with Cy3-dUTP in mid– or late S phase (50 or 90 min). Cy3-dUTP labeling revealed the distribution of replication foci, each of which is presumed to consist of one or a small number of replicon clusters (Fig. 7, a–c). We next quantified the number of replication foci present under different Cdk levels and the mean Cy3 intensity of individual foci (which provides an indication of the number of replication forks that they contain). Surprisingly, treatment of extracts with up to 10 µM roscovitine predominantly reduced the total number of foci, leaving their intensity largely unchanged (Fig. 7, d and e). Higher concentrations of roscovitine, which more strongly suppressed both Cdk activity and total DNA replication (Fig. 4, a and b), inhibited both foci number and intensity (Fig. 7, d and e). Conversely, stimulating DNA replication with cyclin A increased the number of replication foci. This effect is highly reproducible (Fig. S5 a) and is also seen when replication factories are visualized with an anti-PCNA antibody (Fig. S5, b–e). Consistent with these results, DNA fiber analysis showed that treatment of extracts with 10 µM roscovitine did not significantly change either replication fork speed or the density of replication forks within active replicon clusters (Fig. 8). These experiments show that modest changes to Cdk activity preferentially alter the activation of replication factories without significantly changing the rate of initiation within active factories.


Replication factory activation can be decoupled from the replication timing program by modulating Cdk levels.

Thomson AM, Gillespie PJ, Blow JJ - J. Cell Biol. (2010)

Replication foci number depends on Cdk levels. CHOC-400 nuclei were incubated in X. laevis egg extracts incubated at 10,000 nuclei/µl in egg extracts supplemented with the indicated concentrations of roscovitine (rosc) or cyclin A (cyc A). Parallel incubations were supplemented with α-[32P]dATP to measure total DNA synthesis. At 50 or 90 min, extract was pulsed for 5 min with Cy3-dUTP. (a–c) Representative images from the 50-min time point are shown. (d and e) The number and mean intensity of Cy3-labeled foci at 50 (d) and 90 (e) min were measured. Error bars indicate SEM between different nuclei.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812520&req=5

fig7: Replication foci number depends on Cdk levels. CHOC-400 nuclei were incubated in X. laevis egg extracts incubated at 10,000 nuclei/µl in egg extracts supplemented with the indicated concentrations of roscovitine (rosc) or cyclin A (cyc A). Parallel incubations were supplemented with α-[32P]dATP to measure total DNA synthesis. At 50 or 90 min, extract was pulsed for 5 min with Cy3-dUTP. (a–c) Representative images from the 50-min time point are shown. (d and e) The number and mean intensity of Cy3-labeled foci at 50 (d) and 90 (e) min were measured. Error bars indicate SEM between different nuclei.
Mentions: When progression into a new stage of the timing program occurs, new initiation events must occur in newly activated replication factories. Therefore, we investigated which of these aspects were most strongly affected when Cdk activity was varied. CHO nuclei replicating in vitro plus or minus roscovitine or cyclin A were pulsed with Cy3-dUTP in mid– or late S phase (50 or 90 min). Cy3-dUTP labeling revealed the distribution of replication foci, each of which is presumed to consist of one or a small number of replicon clusters (Fig. 7, a–c). We next quantified the number of replication foci present under different Cdk levels and the mean Cy3 intensity of individual foci (which provides an indication of the number of replication forks that they contain). Surprisingly, treatment of extracts with up to 10 µM roscovitine predominantly reduced the total number of foci, leaving their intensity largely unchanged (Fig. 7, d and e). Higher concentrations of roscovitine, which more strongly suppressed both Cdk activity and total DNA replication (Fig. 4, a and b), inhibited both foci number and intensity (Fig. 7, d and e). Conversely, stimulating DNA replication with cyclin A increased the number of replication foci. This effect is highly reproducible (Fig. S5 a) and is also seen when replication factories are visualized with an anti-PCNA antibody (Fig. S5, b–e). Consistent with these results, DNA fiber analysis showed that treatment of extracts with 10 µM roscovitine did not significantly change either replication fork speed or the density of replication forks within active replicon clusters (Fig. 8). These experiments show that modest changes to Cdk activity preferentially alter the activation of replication factories without significantly changing the rate of initiation within active factories.

Bottom Line: We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei.Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it.This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
In the metazoan replication timing program, clusters of replication origins located in different subchromosomal domains fire at different times during S phase. We have used Xenopus laevis egg extracts to drive an accelerated replication timing program in mammalian nuclei. Although replicative stress caused checkpoint-induced slowing of the timing program, inhibition of checkpoint kinases in an unperturbed S phase did not accelerate it. Lowering cyclin-dependent kinase (Cdk) activity slowed both replication rate and progression through the timing program, whereas raising Cdk activity increased them. Surprisingly, modest alteration of Cdk activity changed the amount of DNA synthesized during different stages of the timing program. This was associated with a change in the number of active replication factories, whereas the distribution of origins within active factories remained relatively normal. The ability of Cdks to differentially effect replication initiation, factory activation, and progression through the timing program provides new insights into the way that chromosomal DNA replication is organized during S phase.

Show MeSH
Related in: MedlinePlus