Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.
On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, γ-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy’s laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.
1. LSE Preparation
2. Treatment of Sperm Chromatin with DNA Damaging Approaches
3. Preparation of a DNA Damage-mimicking Structure (AT70)
4. Triggering the DNA Damage Checkpoint in LSE with Damaged Sperm Chromatin or a DNA Damage-mimicking Structure
5. Representative Results
The damaged sperm chromatin or DNA damage-mimicking structure can trigger the ATR/Chk1-mediated DNA damage checkpoint in the Xenopus egg extract system. Figure 2A shows that MMS induces Chk1 phosphorylation at Ser344 (Chk1 P-S344), which is an indicator of ATR kinase activation. Figure 2B shows that AT70, as a DNA damage-mimicking structure, also triggers Chk1 phosphorylation. Total Chk1 samples are used as loading controls in both examples.
Figure 1. A diagram of the DNA damage checkpoint signaling.
Figure 2. Chk1 phosphorylation is induced by either MMS or AT70 treatments in Xenopus egg extracts. (A) MMS-damaged sperm chromatin (MMS) or normal sperm chromatin (Con) are incubated in egg extracts for 90 min. Chk1 phosphorylation at Ser344 (Chk1 P-S344) and total Chk1 in egg extracts are examined via immunoblotting. (B) AT70 or water (Con) are added into egg extracts, respectively. Samples are also analyzed via immunoblotting as in (A).
There are several advantages in studying the DNA damage checkpoint using Xenopus egg extracts. The use of egg extracts provides a large quantity of cell-free extracts synchronized at interphase of the cell cycle. The egg extracts can be easily and inexpensively made. It is relatively easy to damage DNA or chromatin and to reveal a defect in the DNA damage checkpoint after immunodepleting a target protein from egg extract. Subsequently, a potential function defect can be “rescued” by addback of wild type or mutant recombinant proteins. Therefore, Xenopus egg extract is a powerful model system for structure and function analysis of DNA damage checkpoint.
The LSE preparation has been demonstrated previously14, but our protocol has some modifications. The temperature of incubating frogs after hCG injection (18 °C) is very crucial for egg laying. More resources of handling and injecting frogs can be found from previous studies13,14 and on the internet such as tropicalis.berkeley.edu/home/obtaining_embryos/hcg/hCG.html. Generally, 1-3 ml of LSE can be prepared from eggs derived from one frog. It takes approximately one hour to prepare the LSE and it is typically stable on ice for around 4 hours. LSE has to be made fresh when it is needed since freezing and thawing compromises the quality of the LSE for use in DNA damage checkpoint experiments. In Step 1.9, we recommend making stock solutions, saving aliquots in freezers, and using frozen aliquoted stocks for each chemical. In our experience, these aliquots are usable for up to 3 freeze-thaw cycles.
DNA damaging reagents such as MMS have been examined in LSE for triggering Chk1 phosphorylation (see Figure 2A). DNA damaging reagents can either be added directly to LSE or used to damage sperm chromatin before addition to LSE. The concentration of DNA damaging reagents will most likely need to be optimized for specific experiments in order to obtain the best conditions to trigger Chk1 phosphorylation. UV irradiation can also be utilized to damage sperm chromatin and study activation of the DNA damage checkpoint via Chk1 phosphorylation (data not shown). The Chk1 phosphorylation site Ser344 in Xenopus is the conserved site analogous to Chk1 Ser345 in humans 8.
The synthetic oligo AT70 mixture mimics the structure of DNA damage. In the presence of Tautomycin (a phosphatase inhibitor), AT70-induced Chk1 phosphorylation is stabilized and can be examined via immunoblotting (as shown in Figure 2B). The total endogenous Chk1 is used as a loading control. AT70 triggers a mobility shift in total Chk1 that can be visualized on a Western blot, indicating Chk1 is phosphorylated. Antibodies against Chk1 phosphorylation and total Chk1 are suitable for immunoblotting analysis of DNA damage checkpoint signaling in Xenopus.
Overall, the Xenopus egg extracts system is a powerful cell-free model system to investigate the DNA damage checkpoint. This protocol provides the detailed procedures for such analysis. This system can be modulated to study the molecular mechanisms of the DNA damage checkpoint using a variety of different DNA damaging approaches.
The authors have nothing to disclose.
This work is supported in part by funds provided by The University of North Carolina at Charlotte, Wachovia foundation fund for faculty excellence, and a grant from NIGMS (R15GM101571).
Reagents | |||
Anti-Chk1 P-S344 antibody | Cell Signaling | 2348L | |
Anti-Chk1 antibody | Santa Cruz | SC7898 | |
Aprotinin | MP Biomedicals | 0219115880 | |
Cycloheximide | Sigma | C7698-5G | |
Cytochalasin B | EMD | 250233 | |
Dithiothreitol (DTT) | VWR | JTF780-2 | |
hCG | Sigma | CG10-10VL | |
L-Cysteine | Sigma | C7352-1KG | |
Leupeptin | VWR | 97063-922 | |
Methyl methanesulfonate (MMS) | Sigma | 129925-5G | |
Nocodazole | Sigma | M1404-2MG | |
PMSG | Calbiochem | 367222 | |
Sample buffer | Sigma | S3401 | |
Tautomycin | Wako Chemicals USA | 209-12041 | |
Equipment | |||
Bucket for egg laying | Rubbermaid commercial products | 6308 | |
CL2 IEC centrifuge with swinging bucket rotor | Thermo Scientific | 004260F | |
HB6 swinging bucket rotor | Thermo Scientific | 11860 | |
Sorvall RC6 plus superspeed centrifuge | Thermo Scientific | 46910 | |
UV crosslinker | UVP | 95-0174-01 | |
Solutions | |||
1x MMR | 100 mM NaCl, 2 mM KCl, 0.5 mM MgSO4, 2.5 mM CaCl2, 5 mM HEPES, adjust pH to 7.8 with 10 M NaOH | ||
Aprotinin/Leupeptin stock | 10 mg/ml each in water. Store 20 μl aliquots at -80 °C. | ||
Buffer X | 0.2 M sucrose, 80 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 10 mM HEPES, adjust pH to 7.5 by HCl | ||
Cycloheximide stock | 10 mg/ml in water. Store 1 ml aliquots at -20 °C. | ||
Cytochalasin B stock | 5 mg/ml in DMSO. Store 20 μl aliquots at -20 °C. | ||
Dithiothreitol (DTT) stock | 1 M in water. Store 1 ml aliquots at -20 °C. | ||
ELB | 0.25 M sucrose, 1 mM DTT, 50 μg/ml cycloheximide, 2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES, pH7.7 | ||
Nocodazole stock | 10 mg/ml in DMSO. Store 5 μl aliquots at -80 °C. | ||
Energy Mixture | 375 mM creatine phosphate, 50 mM ATP, and 25 mM MgCl2. Aliquots are saved at -80 °C. | ||
Nuclear dye solution | 0.4 μg/ml Hoechst 33258, 25% glycerol (v/v), in 1x PBS | ||
Tautomycin stock | 100 μM in DMSO. Store 10 μl aliquots at -80 °C. |