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	Figure 1. Extracts deficient in RPA chromatin hyperloading are checkpoint proficient. (A) Western blot of chromatin fractions obtained upon incubation of untreated (−) or UV-irradiated (+) sperm chromatin into replication-competent (LSS) or replication-incompetent (HSS) egg extracts for 60 min. (B, inset) Western blot of control egg supernatants, or extracts containing a low level of RPA (RPALow see ‘Materials and Methods’ section). (B) Kinetics of DNA synthesis of control extracts, or extracts reconstituted with a low amount of RPA (RPALow). Aphidicolin was added 80 min after incubation at room temperature (arrow). (C) Western blot of chromatin fractions obtained from the experiment described in (B) in the presence (+) or absence (−) of aphidicolin, or UV-irradiated sperm chromatin. (D) Western blot of nucleoplasmic soluble fractions obtained from the experiment described in (B) in the presence (+) or absence (−) of aphidicolin, or UV irradiation.
	Figure 2. Activation of the replication checkpoint with limited amounts of a recombinant RPA complex. (A, inset) Western blot of egg supernatants after depletion with control (ΔMock) or RPA-specific antibodies (ΔRPA32). (A) Kinetics of DNA synthesis of egg extracts depleted with either control antibodies (ΔMock), or RPA antibodies (ΔRPA) reconstituted with a recombinant RPA complex (ΔRPA + Rec RPA). (B) Silver stain of the RPA recombinant complex. Arrows indicate the three RPA subunits. (C) Western blot of chromatin fraction obtained upon incubation of sperm chromatin in egg extracts depleted with RPA antibodies (A) in the absence (−) or presence (+) of aphidicolin and reconstituted with low or high amounts of recombinant RPA complex (Rec RPA). (D) Analysis of Chk1 phosphorylation in nuclear soluble fractions of the experiment described in panel (C). (E) Quantification of the level of RPA accumulation onto chromatin of the experiment described in (C). Western blot signals were quantified by densitometry scanning and expressed as relative optical density (ROD) compared to the histone H3 signal as loading control. (F) Quantification of the level of Chk1-PS344 in nuclear soluble fractions of the experiment described in (D). Western blot signals were quantified and expressed as relative optical density (ROD) compared to the MCM3 signal that serves here as loading control.
	Figure 3. Chk1 phosphorylation in the absence of RPA hyperloading is sensitive to both caffeine and mitomycin C. (A) Level of Chk1 phosphorylation in control extracts, or extracts containing a low level of RPA (RPALow) in the presence or absence of the indicated compounds, obtained by quantification of western blot signals obtained with the anti Chk1-PS344 antibody. (B) Western blot of chromatin fractions obtained in control extracts, or extracts containing reduced levels of RPA (RPALow), with (+) or without (−) aphidicolin. (C) Schematic diagram of aphidicolin and mitomycin C effects on DNA unwinding during a replication arrest. DNA pols indicates replicative DNA polymerases.
	Figure 4. Spontaneous checkpoint activation in extracts containing a low level of RPA. (A) Western blot of egg supernatants mock-depleted (ΔMock) or RPA partially depleted (ΔRPA32Par). RPA70 indicates the 70-kDa subunit of the RPA complex codepleted by the RPA32 antibody. (B) Western blot of chromatin fractions obtained upon incubation of demembranated sperm chromatin in control extract (ΔMock) or extracts partially-depleted with the RPA32 antibody (ΔRPA32Par) in the absence (−) or presence (+) of apidicolin. (C) Detection of phosphorylated Chk1 (Chk1 P-S344) in nucleosolic fractions obtained upon detergent extraction of nuclei of the experiment described in panel B. (D, upper panel) Western blot of egg supernatants mock-depleted (ΔMock) or RPA partially-depleted (ΔRPA32Par1 or ΔRPA32Par2). (Lower panel) Detection of Chk1 phosphorylation by western blot with anti-phospho Chk1 P-S344 antibody with (+) or without (−) aphidicolin in mock-depleted (ΔMock) or RPA partially-depleted extracts (ΔRPA32Par1 or ΔRPA32Par2). (E) Kinetics of DNA synthesis of mock-depleted (ΔMock) or RPA partially depleted extracts (ΔRPAPar1 or ΔRPA32Par2). (Inset) Autoradiography of nascent DNA obtained by incubation of sperm chromatin in control (ΔMock) or RPA partially-depleted egg extracts (ΔRPA32Par1). (F) Detection of ssDNA in RPA partially-depleted extracts. BrdU-substituted nuclei, prepared as described in Materials and methods, were incubated in control (ΔMock) or RPA partially-depleted extracts (ΔRPAPar2) for 2 h. Nuclei were purified and ssDNA was detected by indirect immunofluorescence with an anti-BrdU antibody by omitting the denaturation step. (G) Kinetics of DNA synthesis of egg extracts in the absence or presence of Geminin. (H) Western blot of chromatin or nuclear fractions in the absence (−) or presence (+) of geminin. A sample of extracts treated with aphidicolin (+Aphi) is included as a positive control. (I, upper panel) Western blot of egg extracts depleted with control antibodies (ΔMock) or MCM8-specific antibodies (ΔMCM8). (Lower panel) Detection of Chk1 P-S344 kinase by western blot in egg extracts depleted with control antibodies (ΔMock) or MCM8-specific antibodies (ΔMCM8) in the absence (−) or presence (+) of aphidicolin.

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