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First published online 9 November 2004
doi: 10.1242/jcs.01400

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

M. Gloria Luciani^*, Maren Oehlmann and J. Julian Blow

Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (43K): [in a new window]   Fig. 1. Replication kinetics in Xenopus egg extract. (A) Sperm nuclei were incubated at 12 ng DNA µl-1 in interphase Xenopus egg extract supplemented with [-32P]dATP. At the indicated times, total DNA synthesis was measured. (B) Sperm nuclei were incubated in extract at 12 ng DNA µl-1. At the indicated times, samples were pulse labelled with [-32P]dATP for 2 minutes. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled -phage HindIII-digested DNA is also shown.

View larger version (85K): [in a new window]   Fig. 2. Inhibition of CDK activity prevents later origins from firing. (A) Sperm nuclei were incubated at 15 ng DNA µl-1 in egg extract supplemented with [-32P]dATP. Aliquots were supplemented with 0.5 mM roscovitine at 20, 25, 30, 35, 40 and 50 minutes. 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-1 in egg extract supplemented with biotin-dUTP. 0.5 mM roscovitine was added at the indicated times. At 120 minutes, nuclei were isolated and stained with Texas Red streptavidin to reveal nuclei that had undergone DNA replication. The proportion of biotin-positive nuclei for each point is shown. (C,D) Sperm nuclei were incubated at 15 ng DNA µl-1 in egg extract. At 35 minutes (C) or 45 minutes (D), aliquots were supplemented with 0.5 mM roscovitine. Samples were pulse labelled with [-32P]dATP for 2 minutes at the indicated times. DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled -phage HindIII-digested DNA is also shown.

View larger version (68K): [in a new window]   Fig. 3. Aphidicolin induces a caffeine-sensitive replication checkpoint. (A-D) Sperm nuclei were incubated at 15 ng DNA µl-1 in interphase egg extract supplemented with [-32P]dATP plus various concentrations of aphidicolin plus or minus 5 mM caffeine. (A) Total DNA synthesis was measured at 120 minutes. (B) DNA synthesis was measured between 15 minutes and 120 minutes as indicated. (C,D) At 120 minutes, DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled -phage HindIII-digested DNA is also shown. (E,F) Sperm nuclei were incubated at 15 ng DNA µl-1 for 35 minutes in interphase egg extract supplemented with 7.5 µM 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 [-32P]dATP and then DNA was separated on an alkaline agarose gel and autoradiographed. The migration of end-labelled -phage HindIII-digested DNA is also shown.

View larger version (38K): [in a new window]   Fig. 4. The intra-S-phase checkpoint reduces the number of active replication forks. (A,B) Sperm nuclei were incubated at 10 ng DNA µl-1 in interphase extract with or without 7.5 µM or 15 µM aphidicolin, minus (A) or plus (B) 5 mM caffeine. During early S phase (35 minutes), nuclei were isolated and transferred to fresh extract supplemented with [-32P]dATP and 0.5 mM roscovitine. Total DNA synthesis was measured at the indicated times after transfer (1-30 minutes). (C,D) Sperm nuclei were incubated in interphase extract at 10 ng DNA µl-1 supplemented with 40 µM aphidicolin minus (C) or plus (D) caffeine. Sperm chromatin was incubated for 35 minutes, 45 minutes or 60 minutes and was then isolated and transferred to fresh extract supplemented with [-32P]dATP and 0.5 mM roscovitine. Total DNA synthesis was measured at different times after transfer.

View larger version (77K): [in a new window]   Fig. 5. The intra-S-phase checkpoint reduces Cdc45 and PCNA on chromatin. (A) Sperm nuclei were incubated at 10 ng DNA µl-1 in interphase extract supplemented with various concentrations of aphidicolin minus (left) or plus (right) 5 mM caffeine. Chromatin was isolated and immunoblotted for XCdc45 and Rad17. (B) Sperm nuclei were incubated at 10 ng DNA µl-1 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.

View larger version (50K): [in a new window]   Fig. 6. Fork stability is not significantly affected by the intra-S-phase checkpoint. (A-C) Sperm nuclei were incubated at 10 ng DNA µl-1 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 minutes, 10 minutes, 25 minutes or 55 minutes after this, nuclei were isolated and transferred to fresh extract supplemented with 0.5 mM roscovitine and [-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-1 in extract. At 35 minutes, 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 [-32P]dATP for 2 minutes, the DNA was isolated and analysed by agarose electrophoresis and autoradiography. The migration of end-labelled -phage HindIII-digested DNA is also shown.

View larger version (22K): [in a new window]   Fig. 7. The ability of caffeine to rescue replication time decreases with time. (A) Sperm nuclei were incubated at 15 ng DNA µl-1 in extract supplemented with [-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 minutes, when total DNA synthesis was measured. As a control, aphidicolin was substituted with DMSO. (B) Sperm nuclei were incubated at 15 ng DNA µl-1 in extract supplemented with [-32P]dATP and 10 µM aphidicolin, plus or minus 5 mM caffeine and increasing concentrations of His6-N-Cyclin A (Ncyclin A). At 120 minutes total DNA synthesis was measured.

View larger version (45K): [in a new window]   Fig. 8. ATR is necessary for the aphidicolin-induced intra-S-phase checkpoint. (A,B) Sperm nuclei were incubated at 15 ng DNA µl-1 in extract supplemented with [-32P]dATP, and various concentrations of aphidicolin, plus or minus 5 mM caffeine and (A) an antibody neutralizing the ATR kinase (-X-ATR) 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-1 in extract supplemented with 0 µM, 15 µM or 120 µM aphidicolin, plus or minus 800 nM wortmannin or 5 mM caffeine. (C) At 50 minutes (mid-S-phase), chromatin was isolated and immunoblotted for Cdc45 and PCNA. (D) At 60 minutes, intact nuclei were isolated and immunoblotted for phospho-Ser344-Chk1. (E) Sperm nuclei were incubated at 15 ng DNA µl-1 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. `?' indicates an unknown cross-reacting protein. (G) Sperm nuclei were incubated at 15 ng DNA µl-1 in Chk1-depleted extract, non-immune-depleted extract or untreated extract, all supplemented with [-32P]dATP and combinations of 10 µM aphidicolin and/or 5 mM caffeine. At 120 minutes, total DNA synthesis was measured.

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