Gillespie PJ , Gambus A , Blow JJ .

083524/Z/07/Z Wellcome Trust , BB/H013024/1 Biotechnology and Biological Sciences Research Council , C303/A7399 Cancer Research UK, 083524 Wellcome Trust , A7999 Cancer Research UK, WT083524 Wellcome Trust , BB_BB/H013024/1 Biotechnology and Biological Sciences Research Council , CRUK_A7999 Cancer Research UK, 14301 Cancer Research UK

	Fig. 1. Xenopus eggs. (A and B) Unactivated, meiosis II metaphase arrested eggs, (C) activated egg and (D) apoptotic egg. A, C and D show top views; B shows side view.
	Fig. 2. Preparation of Xenopus egg extracts. (A) Egg extract, post-crushing spin. (i) lipid layer; (ii) crude cytoplasm; (iii) yolk platelets. Recovery of the crude cytoplasm by side puncture is shown. (B) Egg extract, post-clarifying spin. (i) lipid layer; (ii) cytoplasm; (iii) membranous layer, contains mitochondria; (iv) residual yolk platelets and insoluble material. (C and D) Drop-freezing the final extract in 20 μl aliquots in liquid nitrogen using a cut pipette tip and a plastic Petri dish.
	Fig. 3. Preparation of demembranated Xenopus sperm nuclei. (A) Schematic representation of the body portion of a frog; dashed box indicates the expected location of the testes internally. (B) Testes, in situ, post-incision. ∗testes; ∗∗digestive system. (C and D) Excised testis, pre- (C) and post- (D) removal of extraneous tissue and blood vessels. (E) Chopping the testes in a plastic Petri dish using a razor blade. (F) Filtered sperm solution.
	Fig. 4. Nuclear assembly and DNA replication in Xenopus egg extracts. (A) Timecourse of nuclear formation in Xenopus egg extracts. Sperm nuclei were incubated in metaphase arrested Xenopus egg extract released into interphase by addition of 0.3 mM CaCl2. Nuclear formation was followed over the course of 60 min by phase contrast (upper panels) and UV (lower panels) microscopy (i–vii). Sperm nuclei incubated in extract in the absence of CaCl2 were visualised after 60 min (viii). Bar = 10 μm. (B) The replication kinetics of sperm nuclei added to interphase egg extract as determined by the ‘TCA replication assay’.
	Fig. 5. Timecourse of replication factors association with chromatin. The replication reaction was assembled plus or minus 100 nM CDK inhibitor p27KIP1. (A) DNA synthesis was assayed by α32P-dATP incorporation. (B) Chromatin was isolated at the indicated times, separated by SDS–PAGE and immunoblotted with antibodies against the indicated replication proteins. The lower portion of the protein gel was stained with Coomassie for histones.
	Fig. 6. Release of native proteins from chromatin. Chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin) and proteins were optionally released from chromatin with benzonase. (Ex), 0.5 μl egg extract; (Ch), isolated chromatin, from 5 μl extract, after first centrifugation; other lanes correspond to insoluble pellet (P) and soluble supernatant (S) from 5 μl extract after a second centrifugation. Samples were separated by SDS–PAGE and immunoblotted with antibodies against indicated proteins.
	Fig. 7. Isolated intact nuclei. Nuclei were isolated from egg extract 40 min after replication reaction assembly. (A) Nucleus before isolation. (B) Isolated nucleus. (i) Phase contrast light microscopy; (ii) DNA was stained with Hoechst 33258 and visualised by UV fluorescence microscopy.

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