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DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts.

Gaggioli V, Le Viet B, Germe T, Hyrien O - Nucleic Acids Res. (2013)

Bottom Line: We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes.ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks.Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France.

ABSTRACT
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.

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Alkaline gel electrophoretic analysis of ICRF-193-induced perturbation of DNA synthesis. (A) ICRF-193 added at 0 min, but not 20 min, reduces the abundance and perturbs the growth of nascent DNA strands. Sperm nuclei were incubated in egg extract containing [α-32P]-dATP in the presence of DMSO or 100 µM ICRF-193, added at 0 or at 20 min. Nascent strand abundance and growth was monitored by alkaline gel electrophoresis at the indicated time points. (B) Phosphorimager scan profiles of the 40 min lanes in (A). (C) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in egg extract in the presence of DMSO or ICRF-193 added at 0 min or 20 min as indicated were labeled at 28 min with a 2 min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were analyzed as in (A). (D) Nascent strand maturation analysis. Same experiment as in (A) except for the indicated times.
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gkt494-F3: Alkaline gel electrophoretic analysis of ICRF-193-induced perturbation of DNA synthesis. (A) ICRF-193 added at 0 min, but not 20 min, reduces the abundance and perturbs the growth of nascent DNA strands. Sperm nuclei were incubated in egg extract containing [α-32P]-dATP in the presence of DMSO or 100 µM ICRF-193, added at 0 or at 20 min. Nascent strand abundance and growth was monitored by alkaline gel electrophoresis at the indicated time points. (B) Phosphorimager scan profiles of the 40 min lanes in (A). (C) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in egg extract in the presence of DMSO or ICRF-193 added at 0 min or 20 min as indicated were labeled at 28 min with a 2 min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were analyzed as in (A). (D) Nascent strand maturation analysis. Same experiment as in (A) except for the indicated times.

Mentions: To further understand the mechanism(s) by which ICRF-193 reduces DNA synthesis, sperm nuclei were incubated in extracts with [α-32P]dATP, and nascent DNA strands were analyzed at 25, 30, 35 and 40 min by alkaline agarose gel electrophoresis (Figure 3A and B). ICRF-193 added at 0 min did not delay the start of S phase but reduced the incorporation of radioactive label by ∼40% at all time points. The growth of nascent strands was delayed with respect to the control when ICRF-193 was added at 0 min, but not 20 min. Strand size distributions depend on initiation, elongation and termination rates. Elongation rates were measured by labeling nascent strands at 28 min by a 2-min pulse of [α-32P]dATP, chasing the label with unlabeled dATP in the presence of roscovitin, a cyclin-dependent kinase inhibitor that blocks further initiation, and measuring strand growth between early time points, when replicon merge was minimal (Figure 3C). Elongation rates were found to be 500 nt.min−1 in the control but 400 nt.min−1 when ICRF-193 was added at 0 min. This 20% inhibition did not fully account for the 40% inhibition of [α-32P]dATP incorporation, suggesting that origin firing was also reduced, as shown below by DNA combing.Figure 3.


DNA topoisomerase IIα controls replication origin cluster licensing and firing time in Xenopus egg extracts.

Gaggioli V, Le Viet B, Germe T, Hyrien O - Nucleic Acids Res. (2013)

Alkaline gel electrophoretic analysis of ICRF-193-induced perturbation of DNA synthesis. (A) ICRF-193 added at 0 min, but not 20 min, reduces the abundance and perturbs the growth of nascent DNA strands. Sperm nuclei were incubated in egg extract containing [α-32P]-dATP in the presence of DMSO or 100 µM ICRF-193, added at 0 or at 20 min. Nascent strand abundance and growth was monitored by alkaline gel electrophoresis at the indicated time points. (B) Phosphorimager scan profiles of the 40 min lanes in (A). (C) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in egg extract in the presence of DMSO or ICRF-193 added at 0 min or 20 min as indicated were labeled at 28 min with a 2 min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were analyzed as in (A). (D) Nascent strand maturation analysis. Same experiment as in (A) except for the indicated times.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753627&req=5

gkt494-F3: Alkaline gel electrophoretic analysis of ICRF-193-induced perturbation of DNA synthesis. (A) ICRF-193 added at 0 min, but not 20 min, reduces the abundance and perturbs the growth of nascent DNA strands. Sperm nuclei were incubated in egg extract containing [α-32P]-dATP in the presence of DMSO or 100 µM ICRF-193, added at 0 or at 20 min. Nascent strand abundance and growth was monitored by alkaline gel electrophoresis at the indicated time points. (B) Phosphorimager scan profiles of the 40 min lanes in (A). (C) Pulse-chase analysis of nascent strand growth. Sperm nuclei incubated in egg extract in the presence of DMSO or ICRF-193 added at 0 min or 20 min as indicated were labeled at 28 min with a 2 min pulse of [α-32P]dATP and chased for the indicated times with unlabeled dATP in the presence of roscovitin. Nascent strands were analyzed as in (A). (D) Nascent strand maturation analysis. Same experiment as in (A) except for the indicated times.
Mentions: To further understand the mechanism(s) by which ICRF-193 reduces DNA synthesis, sperm nuclei were incubated in extracts with [α-32P]dATP, and nascent DNA strands were analyzed at 25, 30, 35 and 40 min by alkaline agarose gel electrophoresis (Figure 3A and B). ICRF-193 added at 0 min did not delay the start of S phase but reduced the incorporation of radioactive label by ∼40% at all time points. The growth of nascent strands was delayed with respect to the control when ICRF-193 was added at 0 min, but not 20 min. Strand size distributions depend on initiation, elongation and termination rates. Elongation rates were measured by labeling nascent strands at 28 min by a 2-min pulse of [α-32P]dATP, chasing the label with unlabeled dATP in the presence of roscovitin, a cyclin-dependent kinase inhibitor that blocks further initiation, and measuring strand growth between early time points, when replicon merge was minimal (Figure 3C). Elongation rates were found to be 500 nt.min−1 in the control but 400 nt.min−1 when ICRF-193 was added at 0 min. This 20% inhibition did not fully account for the 40% inhibition of [α-32P]dATP incorporation, suggesting that origin firing was also reduced, as shown below by DNA combing.Figure 3.

Bottom Line: We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes.ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks.Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie de l'Ecole Normale Supérieure (IBENS), S2-Génomique Fonctionnelle, CNRS UMR8197, Inserm U1024, 46 rue d'Ulm, 75005 Paris, France.

ABSTRACT
Sperm chromatin incubated in Xenopus egg extracts undergoes origin licensing and nuclear assembly before DNA replication. We found that depletion of DNA topoisomerase IIα (topo IIα), the sole topo II isozyme of eggs and its inhibition by ICRF-193, which clamps topo IIα around DNA have opposite effects on these processes. ICRF-193 slowed down replication origin cluster activation and fork progression in a checkpoint-independent manner, without altering replicon size. In contrast, topo IIα depletion accelerated origin cluster activation, and topo IIα add-back negated overinitiation. Therefore, topo IIα is not required for DNA replication, but topo IIα clamps slow replication, probably by forming roadblocks. ICRF-193 had no effect on DNA synthesis when added after nuclear assembly, confirming that topo IIα activity is dispensable for replication and revealing that topo IIα clamps formed on replicating DNA do not block replication, presumably because topo IIα acts behind and not in front of forks. Topo IIα depletion increased, and topo IIα addition reduced, chromatin loading of MCM2-7 replicative helicase, whereas ICRF-193 did not affect MCM2-7 loading. Therefore, topo IIα restrains MCM2-7 loading in an ICRF-193-resistant manner during origin licensing, suggesting a model for establishing the sequential firing of origin clusters.

Show MeSH
Related in: MedlinePlus