Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus

Abstract

In most eukaryotes, replication origins fire asynchronously throughout S-phase according to a precise timing programme. When replication fork progression is inhibited, an intra-S-phase checkpoint is activated that blocks further origin firing and stabilizes existing replication forks to prevent them undergoing irreversible collapse. We show that chromatin incubated in Xenopus egg extracts displays a replication-timing programme in which firing of new replication origins during S phase depends on the continued activity of S-phase-inducing cyclin-dependent kinases. We also show that low concentrations of the DNA-polymerase inhibitor aphidicolin, which only slightly slows replication-fork progression, strongly suppress further initiation events. This intra-S-phase checkpoint can be overcome by caffeine, an inhibitor of the ATM/ATR checkpoint kinases, or by neutralizing antibodies to ATR. However, depletion or inhibition of Chk1 did not abolish the checkpoint. We could detect no significant effect on fork stability when this intra-S-phase checkpoint was inhibited. Interestingly, although caffeine could prevent the checkpoint from being activated, it could not rescue replication if added after the timing programme would normally have been executed. This suggests that special mechanisms might be necessary to reverse the effects of the intra-S-phase checkpoint once it has acted on particular origins.

Figure 1

Replication kinetics in Xenopus egg extract. (A) Sperm nuclei were incubated at 12 ng DNA/μl in interphase Xenopus egg extract supplemented with [α-³²P]dATP. At the indicated times, total DNA synthesis was measured. (B) Sperm nuclei were incubated in extract at 12 ng DNA/μl. At the indicated times, samples were pulse labelled with [α-³²P]dATP for 2 min. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-Hind III DNA is also shown.

Figure 2

Inhibition of CDK activity prevents later origins from firing. (A) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with [α-³²P]dATP. Aliquots were supplemented with 0.5 mM roscovitine at the following times: filled triangles, 20 min; filled diamonds, 25 min; filled circles 30 min; open triangles, 35 min; open diamonds, 40 min; open circles, 50 min. Samples with no added roscovitine are shown by open squares. At the indicated times, samples were assayed for total DNA synthesis. (B) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract supplemented with biotin-dUTP. 0.5 mM roscovitine was added at the indicated times. At 120 min, nuclei were isolated and stained with Texas Red streptavidin to reveal nuclei which had undergone DNA replication. The percentage of biotin-positive nuclei for each point is shown. (C, D) Sperm nuclei were incubated at 15 ng DNA/μl in egg extract. At 35 minutes (C) or 45 minutes (D), aliquots were supplemented with 0.5mM roscovitine. Samples were pulse-labelled with [α-³²P]dATP for 2 minutes at the indicated times. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.

Figure 3

Aphidicolin induces a caffeine-sensitive replication checkpoint. (A-D) Sperm nuclei were incubated at 15 ng DNA/μl in interphase egg extract supplemented with [α-³²P]dATP plus various concentrations of aphidicolin plus or minus 5 mM caffeine. (A) Total DNA synthesis was measured at 120 min. (B) DNA synthesis was measured between 15 and 120 minutes as indicated. (C, D) At 120 min, DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown. (E, F) Sperm nuclei were incubated at 15 ng DNA/μl for 35 minutes in interphase egg extract supplemented with 7.5μM aphidicolin. The reaction was split in two, and supplemented with 0.5 mM roscovitine minus (E) or plus (F) 5 mM caffeine. At 5 minute intervals, aliquots were pulse-labelled for 2 minutes with [α-³²P]dATP, then DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled λ-HindIII DNA is also shown.

Figure 4

The intra-S phase checkpoint reduces the number of active replication forks. (A, B) Sperm nuclei were incubated at 10 ng DNA/μl in interphase extract with or without 7.5 μM or 15 μM aphidicolin, minus (A) or plus (B) 5 mM caffeine. At early S-phase (35min), nuclei were isolated and transferred to fresh extract supplemented with [α-³²P]dATP and 0.5 mM roscovitine. At the indicated times after transfer (1-30min), total DNA synthesis was measured. (C, D) Sperm nuclei were incubated in interphase extract at 10 ng DNA/μl supplemented with 40 μM aphidicolin minus (C) or plus (D) caffeine. Sperm chromatin was incubated for 35, 45 or 60 min, and was then isolated and transferred to fresh extract supplemented with [α-³²P]dATP and 0.5 mM roscovitine. Total DNA synthesis was measured at different times after transfer.

Figure 5

The intra-S phase checkpoint reduces Cdc45 and PCNA on chromatin. (A) Sperm nuclei were incubated at 10 ng DNA/μl in interphase extract supplemented with various concentrations of aphidicolin minus (left panel) or plus 5 mM caffeine (right panel). Chromatin was isolated and immunoblotted for XCdc45 and Rad17. (B) Sperm nuclei were incubated at 10 ng DNA/μl in interphase extract supplemented with different combinations of 15 μM aphidicolin and/or 5 mM caffeine. At the indicated times, chromatin was isolated and immunoblotted for XCdc45 and PCNA.

Figure 6

Fork stability is not significantly affected by the intra-S phase checkpoint. (A, B, C) Sperm nuclei were incubated at 10 ng DNA/μl in Xenopus extract; after 35 minutes (early S-phase) extract was supplemented with 40 μM aphidicolin and 0.5 mM roscovitine minus (B) or plus (C) 5 mM caffeine. 0, 10, 25 or 55 minutes after this, nuclei were isolated and transferred to fresh extract supplemented with 0.5 mM roscovitine and [α-³²P]dATP. Total DNA synthesis was measured at different times after transfer. A schematic outline of the experiment is shown in (A). (D) Sperm nuclei were incubated at 10 ng DNA/μl in extract. At 35 min, the extract was supplemented with 7.5 μM aphidicolin and 5 mM caffeine, minus or plus 0.5 mM roscovitine. At the indicated times, samples were pulse-labelled with [α-³²P]dATP for 2 min, the DNA was isolated and analysed by agarose electrophoresis and autoradiography. The migration of end-labelled λ-HindIII DNA is also shown.

Figure 7

The ability of caffeine to rescue replication time decreases with time. (A) Sperm nuclei were incubated at 15 ng DNA/μl in extract supplemented with [α-³²P]dATP and 15 μM aphidicolin. At the indicated times, 5 mM caffeine plus or minus 0.5 mM roscovitine were added to the extract and the samples incubated for a further 120 min, when total DNA synthesis was measured. As control, aphidicolin was substituted with DMSO. (B) Sperm nuclei were incubated at 15 ng DNA/μl in extract supplemented with [α-³²P]dATP, 10 μM aphidicolin, plus or minus 5 mM caffeine and increasing concentrations of ΔNcyclin A. At 120 minutes total DNA synthesis was measured.

Figure 8

ATR is necessary for the aphidicolin-induced intra-S-phase checkpoint. (A, B) Sperm nuclei were incubated at 15 ng DNA/μl in extract supplemented with [α-³²P]dATP, and various concentrations of aphidicolin, plus or minus 5 mM caffeine or (A) an antibody neutralising the ATR kinase (α-XATR), or (B) 800 nM wortmannin. At 120 minutes total DNA synthesis was measured. As control, aphidicolin was substituted with DMSO. (C, D) Sperm nuclei were incubated at 15 ng DNA/μl in extract supplemented with 0, 15 or 120 μM aphidicolin, plus or minus 800nM wortmannin or 5 mM caffeine. (C) At 50 minutes (mid-S-phase) chromatin was isolated and immunoblotted for Cdc45 and PCNA. (D) At 60 min, intact nuclei were isolated and immunoblotted for phospho-Ser³⁴⁴-Chk1. (E) Sperm nuclei were incubated at 15 ng DNA/μl in extract supplemented with 12 μM aphidicolin plus or minus 5 mM caffeine or 5 μM DBH. At 120 minutes total DNA synthesis was measured. (F, G) Extract was immunodepleted with anti-Chk1 antibodies. (F) Immunoblotting of whole nuclei assembled in either untreated extract, mock depleted or Chk1-depleted extracted showed the removal of Chk1; ‘?’ marks an unknown cross-reacting protein. (G) Sperm nuclei were incubated at 15 ng DNA/μl in Chk1-depleted extract, non-immune-depleted extract or untreated extract, all supplemented with [α-³²P]dATP and combinations of 10 μM aphidicolin and/or 5mM caffeine. At 120 minutes total DNA synthesis was measured.