Chromosomal DNA Replication in a Soluble Cell Free System Derived From Xenopus Eggs

Antonin V. Tutter and Johannes C. Walter

Summary

Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with clarified egg cytoplasm to form chromatin-bound prereplication complexes. Subsequently, a highly concentrated nucleoplasmic extract is added that stimulates initiation from these prereplication complexes, and a single complete round of chromosomal DNA replication ensues. This review describes the preparation of the cytosolic and nucleo-plasmic extracts, as well as their use in DNA replication, origin unwinding, and chromatin isolation assays.

Key Words: Cell-free system; DNA replication; geminin; MCM2-7; nucleoplasmic extract; ORC; origin unwinding; prereplication complex; Xenopus laevis.

1. Introduction

Cell-free extracts made from the eggs of the frog Xenopus laevis represent a powerful system to study the biochemical mechanisms underlying eukaryotic DNA replication. Addition of demembranated sperm chromatin to cytoplasmic extracts of unfertilized eggs leads to the formation of nuclei that undergo a single, complete round of semiconservative DNA replication per cell cycle (1-4). The use of these "nuclear assembly" extracts has led to many fundamental insights into the mechanism and regulation of eukaryotic DNA replication (5).

However, nuclear assembly extracts have several drawbacks. First, any manipulations that interfere with the delicate process of nuclear assembly indirectly affect DNA replication because of defects in nuclear transport, a frequent source of artifacts. Second, although nuclear assembly extracts support DNA replication of purified DNA molecules such as plasmids, the efficiency is very low (1,4). Third, the requirement

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

Fig. 1. DNA replication in nucleus-free extracts. (A) Schematic representation of the nucleus-free DNA replication assay. Sperm chromatin or plasmid DNA is incubated in HSE to promote the assembly of prereplication complexes (pre-RCs). Addition of NPE after pre-RC assembly converts the pre-RCs to active replication forks, and replication commences. NPE also contains inhibitors that prevent de novo pre-RC assembly as well as origin refiring and thus restricts replication to a single round. (B-D) Photos of various centrifugation fractions during the production of LSE, HSE, and NPE, respectively.

Fig. 1. DNA replication in nucleus-free extracts. (A) Schematic representation of the nucleus-free DNA replication assay. Sperm chromatin or plasmid DNA is incubated in HSE to promote the assembly of prereplication complexes (pre-RCs). Addition of NPE after pre-RC assembly converts the pre-RCs to active replication forks, and replication commences. NPE also contains inhibitors that prevent de novo pre-RC assembly as well as origin refiring and thus restricts replication to a single round. (B-D) Photos of various centrifugation fractions during the production of LSE, HSE, and NPE, respectively.

for a nuclear envelope to initiate DNA replication precludes biochemical fractionation of the factors required for these steps.

In this review, we describe a procedure for DNA replication in Xenopus egg extracts that circumvents the need for nuclear assembly (6). In this approach, DNA replication is carried out using two extracts, both of which are derived from low-speed extract (LSE) of egg cytoplasm (Fig. 1A). To make the first extract, LSE is centrifuged at high g-force to generate clarified egg cytosol or high-speed extract (HSE). To make the second extract, LSE is supplemented with sperm chromatin to assemble nuclei on a large scale. The nuclei are harvested and centrifuged at high g-force to separate the soluble nucleoplasmic extract (NPE) from the chromatin and nuclear envelopes.

To initiate DNA replication, DNA templates are first incubated with HSE to form the prereplication complex (pre-RC). This reaction does not require a specific DNA sequence and involves the sequential loading of the initiation factors ORC, Cdc6, Cdtl, and MCM2-7. To initiate DNA replication from pre-RCs, two vol of NPE are added. NPE supplies high concentrations of Cdk2/cyclin E (7), Cdc7/Dbf4 (8), MCM10 (9), and possibly other factors. DNA replication initiates, and a single complete round of DNA replication takes place during which MCM2-7 is lost from chromatin (6). Additional rounds of DNA replication in the nucleus-free system are inhibited because NPE blocks the de novo loading of MCM2-7 (6) to high concentrations of the inhibitor geminin (ref. 10 and our unpublished results).

The nucleus-free system has several advantages over nuclear assembly extracts. First, the system can be used to resolve whether a particular protein is required for DNA replication directly rather than indirectly because of involvements in nuclear envelope assembly (11). Second, in this system, the nuclear environment can be extensively manipulated. For example, different DNA templates can be replicated simultaneously in the same biochemical environment, a feature that was used to demonstrate the existence of diffusible checkpoint signals (12). Third, the nucleus-free system supports efficient DNA replication of small plasmids, allowing structural analysis of key replication events such as origin unwinding (13). In addition, the effects of DNA sequence on DNA replication can be examined. Finally, the demonstration that DNA replication can take place in a completely soluble environment holds the promise that this process can eventually be reconstituted from purified components. Although the current preparation method for NPE is too cost and labor intensive to allow fractionation, NPE may ultimately be replaced with purified components.

In this review, we describe the protocols that are necessary to carry out DNA replication in the nucleus-free system. We first describe how to isolate Xenopus sperm chromatin (Subheading 3.1.). We then describe how to induce egg laying in female frogs (Subheading 3.2.) and the preparation of LSE (Subheading 3.3.), HSE (Subheading 3.4.), and NPE extracts (Subheading 3.5.). Finally, we present protocols for measuring DNA replication (Subheading 3.6.), origin unwinding (Subheading 3.7.), and chromatin loading in the nucleus-free system (Subheading 3.8.); we describe how to use immunodepletion to remove specific proteins from the system to assess their roles in DNA replication (Subheading 3.9.).

2. Materials

2. Aphidicolin (cat. no. A0781; Sigma): dissolve in dimethylsulfoxide (DMSO) to 5 mg/mL. Store 5-|lL one-use aliquots at -70°C.

3. Adenosine triphosphate (ATP; cat. no. A-7699 disodium salt; Sigma): dissolve in water and adjust to pH 7.0 with NaOH. Prepare as a 0.2 M stock and store 50-|L and 1-mL aliquots at -20°C.

4. ATP regeneration mix: combine 10 |L of 1 Mphosphocreatine (PC), 5 |L of 0.2 M ATP, and 0.5 ||L of 5 mg/mL creatine phosphokinase (CK). Prepare fresh and keep on ice before use.

5. Benzocaine (cat. no. E-1501; Sigma): prepare a 10% solution in ethanol at room temperature on the day of use.

6. Bovine serum albumin (BSA; cat. no A7906-100G; Sigma).

7. Buffer X: 10 mM HEPES, pH 7.4, 80 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA). Prepare as a 10X stock and filter sterilize.

8. Chloroquine phosphate (cat. no. C-6628; Sigma): dissolve in water to 40 mM final concentration. Prepare fresh on the day of use.

9. CK (35,000 U; cat. no. C-3755; Sigma): dissolve in 50 mM NaCl, 50% glycerol, 10 mM HEPES, pH 7.5. Prepare as a 5 mg/mL stock and store as 1-mL aliquots at -20°C for up to several months.

10. Cycloheximide (cat. no. 239763; Calbiochem): dissolve in water to 10 mg/mL. Prepare fresh at 4°C on the day of use.

11. Cysteine HCL (cat. no. CY115; Spectrum): prepare a 2.2% solution in room temperature water immediately before use and adjust to pH 7.7 with KOH.

12. Cytochalasin B (cat. no. 6762; Sigma): dissolve in DMSO to 5 mg/mL. Store 50-|lL aliquots at -20°C.

13. 10X ELB (egg lysis buffer) salts: 25 mM MgCl2, 500 mM KCl, 100 mM HEPES. Adjust pH to 7.7 with KOH, filter sterilize, and store at 4°C.

14. ELB: 1 mM dithiothreitol (DTT), 50 |g/mL cycloheximide, 0.25 M sucrose, 1X ELB salts. Prepare at room temperature on the day of use.

15. Ethanol.

17. hCG (human chorionic gonadotropin): 10,000 U (cat. no. CG010; Sigma).

18. Hoechstfix solution: 8 |g/mL Hoechst, 7.4% formaldehyde, 200 mM sucrose, 10 mM HEPES, pH 7.6. Store at room temperature.

19. MMR (Marc's modified Ringer's): 100 mM NaCl, 2 mM KCl, 0.5 mM MgSO4, 2.5 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8. Prepare as a 10X stock and store at 4°C.

20. NaOAc: prepare a 3 M stock and adjust pH to 5.8. Autoclave and store at room temperature.

21. Hypodermic needles (21, 27 gage).

22. Nocodozole (cat. no. M1404; Sigma): dissolve in DMSO to 5-mg/mL and 0.5-mg/mL concentrations. Store 50-|L aliquots of each concentration at -20°C.

23. PC (cat. no. P-6502; Sigma; disodium salt, hydrate, synthetic): dissolve in 10 mM sodium phosphate, pH 7.0. Prepare as a 1 M stock and store 50- and 1-mL aliquots at -20°C.

24. Phenol:chloroform (1/1).

25. Pregnant mare serum gonadotropin (PMSG; 5000 U; cat. no. 367222; Calbiochem).

26. Protease inhibitor mix (Aprotinin; cat. no. 981532; Roche; Leupeptin; cat. no. 1034626; Roche): dissolve each part of the mix in water to 10 mg/mL (1000X stock). Freeze 25-|L aliquots in liquid N2 and store at -70°C.

27. Proteinase K (cat. no. 3115879; Roche): prepare a 20-mg/mL stock in water. Store 50-|L aliquots at -80°C.

28. Replication stop solution: 8 mM EDTA, 0.13% phosphoric acid, 10% Ficoll, 5% sodium dodecyl sulfate (SDS), 0.2% bromophenol blue, 80 mM Tris-HCl, pH 8.0. Store at room temperature.

29. Ribonuclease A (cat. no. R-4875; Sigma): prepare a 2-mg/mL stock in water. Store 50-|L aliquots at -20°C.

30. SDS sample buffer (2X): 50 mM Tris-HCl, pH 6.8, 2% SDS, 100 mM DTT, 10% glycerol, 0.1% bromophenol blue. Store at room temperature for up to 1 wk.

31. Sybergold (cat. no. S-11494; Molecular Probes).

33. 6X TBE loading dye: 50 mM Tris-HCl, pH 7.9, 50% glycerol, 30 mM EDTA, 0.25% bromophenol blue. Store at room temperature for up to 1 mo.

35. Unwinding stop solution: 1% SDS, 20 mM EDTA. Store at room temperature for up to 1 wk.

36. Xenopus laevis females (cat. no. LM00531M; Nasco).

37. Xenopus laevis males (cat. no. LM00713M; Nasco).

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