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).

1. LSE Preparation
<ol>
 <li>Female frogs (Xenopus laevis) are injected twice for egg collection. The first injection (priming) is 100 U PMSG (Pregnant Mare Serum Gonadotropin) per frog. Frogs must be primed at least two days before inducing egg laying and primed frogs are usable for up to two weeks. To prime frogs, inject PMSG subcutaneously in the dorsal lymph sacs using a 3 ml syringe and 27 G needle.</li>
 <li>To induce egg laying, inject 500 U hCG (human Chorionic Gonadotrophin) per primed frog subcutane...

<ol>
	<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220 (4598), 719-721 (1983).</li><li>Cimprich, K. A., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATR:+an+essential+regulator+of+genome+integrity.">ATR: an essential regulator of genome integrity.</a> Nat. Rev. Mol. Cell Biol. 9 (8), 616-627 (2008).</li><li>Lupardus, P. J., Van, C., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+the+ATR-mediated+checkpoint+using+Xenopus+egg+extracts.">Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.</a> Methods. 41 (2), 222-231 (2007).</li><li>Lupardus, P. J., Byun, T., Yee, M. C., Hekmat-Nejad, M., Cimprich, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+requirement+for+replication+in+activation+of+the+ATR-dependent+DNA+damage+checkpoint.">A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.</a> Genes Dev. 16 (18), 2327-2332 (2002).</li><li>Stokes, M. P., Van Hatten, R., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+is+required+for+the+checkpoint+response+to+damaged+DNA+in+Xenopus+egg+extracts.">DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.</a> J. Cell Biol. 158 (5), 863-872 (2002).</li><li>Kato, K., Strauss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+an+intermediate+in+DNA+synthesis+by+HEp.2+cells+treated+with+methyl+methanesulfonate.">Accumulation of an intermediate in DNA synthesis by HEp.2 cells treated with methyl methanesulfonate.</a> Proc. Natl. Acad. Sci. U.S.A. 71 (5), 1969-1973 (1974).</li><li>Paulovich, A. G., Hartwell, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+checkpoint+regulates+the+rate+of+progression+through+S+phase+in+S.+cerevisiae+in+response+to+DNA+damage.">A checkpoint regulates the rate of progression through S phase in S. cerevisiae in response to DNA damage.</a> Cell. 82 (5), 841-847 (1995).</li><li>Guo, Z., Kumagai, A., Wang, S. X., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+Atr+in+phosphorylation+of+Chk1+and+cell+cycle+regulation+in+response+to+DNA+replication+blocks+and+UV-damaged+DNA+in+Xenopus+egg+extracts.">Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.</a> Genes Dev. 14 (21), 2745-2756 (2000).</li><li>Jazayeri, A., Balestrini, A., Garner, E., Haber, J. E., Costanzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mre11-Rad50-Nbs1-dependent+processing+of+DNA+breaks+generates+oligonucleotides+that+stimulate+ATM+activity.">Mre11-Rad50-Nbs1-dependent processing of DNA breaks generates oligonucleotides that stimulate ATM activity.</a> EMBO. J. 27 (14), 1953-1962 (2008).</li><li>Kumagai, A., Dunphy, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claspin,+a+novel+protein+required+for+the+activation+of+Chk1+during+a+DNA+replication+checkpoint+response+in+Xenopus+egg+extracts.">Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.</a> Mol. Cell. 6 (4), 839-849 (2000).</li><li>Yan, S., Lindsay, H. D., Michael, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+requirement+for+Xmus101+in+ATR-mediated+phosphorylation+of+Claspin+bound+Chk1+during+checkpoint+signaling.">Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling.</a> J. Cell Biol. 173 (2), 181-186 (2006).</li><li>Shiotani, B., Zou, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-stranded+DNA+orchestrates+an+ATM-to-ATR+switch+at+DNA+breaks.">Single-stranded DNA orchestrates an ATM-to-ATR switch at DNA breaks.</a> Mol. Cell. 33 (5), 547-558 (2009).</li><li>Tutter, A. V., Walter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+DNA+replication+in+a+soluble+cell-free+system+derived+from+Xenopus+eggs.">Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.</a> Methods Mol. Biol. 322, 121-137 (2006).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Fractionation+of+Xenopus+laevis+Egg+Extracts.">Preparation and Fractionation of Xenopus laevis Egg Extracts.</a> J. Vis. Exp. (18), e891 (2008).</li></ol>

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 muta...

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Anti-Chk1 P-S344 antibody</td>
 <td>Cell Signaling</td>
 <td>2348L</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Chk1 antibody</td>
 <td>Santa Cruz</td>
 <td>SC7898</td>
 <td></td>
 </tr>
 <tr>
 <td>Aprotinin</td>
 <td>MP Biomedicals</td>
 <td>0219115880</td>
 <td></td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>Sigma</td>
 <td>C7698-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>Cytochalasin B</td>
 <td>EMD</td>
 <td>250233</td>
 <td></td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT)</td>
 <td>VWR</td>
 <td>JTF780-2</td>
 <td></td>
 </tr>
 <tr>
 <td>hCG</td>
 <td>Sigma</td>
 <td>CG10-10VL</td>
 <td></td>
 </tr>
 <tr>
 <td>L-Cysteine</td>
 <td>Sigma</td>
 <td>C7352-1KG</td>
 <td></td>
 </tr>
 <tr>
 <td>Leupeptin</td>
 <td>VWR</td>
 <td>97063-922</td>
 <td></td>
 </tr>
 <tr>
 <td>Methyl methanesulfonate (MMS)</td>
 <td>Sigma</td>
 <td>129925-5G</td>
 <td></td>
 </tr>
 <tr>
 <td>Nocodazole</td>
 <td>Sigma</td>
 <td>M1404-2MG</td>
 <td></td>
 </tr>
 <tr>
 <td>PMSG</td>
 <td>Calbiochem</td>
 <td>367222</td>
 <td></td>
 </tr>
 <tr>
 <td>Sample buffer</td>
 <td>Sigma </td>
 <td>S3401</td>
 <td></td>
 </tr>
 <tr>
 <td>Tautomycin</td>
 <td>Wako Chemicals USA</td>
 <td>209-12041</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment </td>
 </tr>
 <tr>
 <td>Bucket for egg laying</td>
 <td>Rubbermaid commercial products</td>
 <td>6308</td>
 <td></td>
 </tr>
 <tr>
 <td>CL2 IEC centrifuge with swinging bucket rotor</td>
 <td>Thermo Scientific</td>
 <td>004260F</td>
 <td></td>
 </tr>
 <tr>
 <td>HB6 swinging bucket rotor</td>
 <td>Thermo Scientific</td>
 <td>11860</td>
 <td></td>
 </tr>
 <tr>
 <td>Sorvall RC6 plus superspeed centrifuge </td>
 <td>Thermo Scientific </td>
 <td>46910</td>
 <td></td>
 </tr>
 <tr>
 <td>UV crosslinker</td>
 <td>UVP</td>
 <td>95-0174-01</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Solutions</td>
 </tr>
 <tr>
 <td>1x MMR</td>
 <td></td>
 <td></td>
 <td>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</td>
 </tr>
 <tr>
 <td>Aprotinin/Leupeptin stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml each in water. Store 20 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 <tr>
 <td>Buffer X</td>
 <td></td>
 <td></td>
 <td>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</td>
 </tr>
 <tr>
 <td>Cycloheximide stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml in water. Store 1 ml aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>Cytochalasin B stock</td>
 <td></td>
 <td></td>
 <td>5 mg/ml in DMSO. Store 20 &mu;l aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>Dithiothreitol (DTT) stock</td>
 <td></td>
 <td></td>
 <td>1 M in water. Store 1 ml aliquots at -20 &deg;C. </td>
 </tr>
 <tr>
 <td>ELB</td>
 <td></td>
 <td></td>
 <td>0.25 M sucrose, 1 mM DTT, 50 &mu;g/ml cycloheximide, 2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES, pH7.7</td>
 </tr>
 <tr>
 <td>Nocodazole stock</td>
 <td></td>
 <td></td>
 <td>10 mg/ml in DMSO. Store 5 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 <tr>
 <td>Energy Mixture</td>
 <td></td>
 <td></td>
 <td>375 mM creatine phosphate, 50 mM ATP, and 25 mM MgCl2. Aliquots are saved at -80 &deg;C.</td>
 </tr>
 <tr>
 <td>Nuclear dye solution</td>
 <td></td>
 <td></td>
 <td>0.4 &mu;g/ml Hoechst 33258, 25% glycerol (v/v), in 1x PBS </td>
 </tr>
 <tr>
 <td>Tautomycin stock</td>
 <td></td>
 <td></td>
 <td>100 &mu;M in DMSO. Store 10 &mu;l aliquots at -80 &deg;C. </td>
 </tr>
 </tbody>
 </table>

study of the dna damage checkpoint using xenopus egg extracts

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, &gamma;-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.

Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

study-of-the-dna-damage-checkpoint-using-xenopus-egg-extracts

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16.5K vues.  University of North Carolina at Charlotte. Xenopus oeuf extrait est un système modèle utile pour étudier le point de contrôle de dommages à l'ADN. Ce protocole est destiné à la préparation d'extraits d'œufs de xénope et de l'ADN réactifs induisant des dommages aux points de contrôle. Ces techniques sont adaptables à une variété d'approches endommageant l'ADN dans l'étude de la signalisation checkpoint dommages de l'ADN.