Name: study of the dna damage checkpoint using xenopus egg extracts
Uploaded: 2012-11-05T13:00:00
Description: Watch this Scientific Journal Video about Study of the DNA Damage Checkpoint using Xenopus Egg Extracts at JoVE.com

इस काम के हिस्से में Charlotte में उत्तरी कैरोलिना विश्वविद्यालय, वाचोविया संकाय उत्कृष्टता के लिए नींव निधि, और NIGMS (R15GM101571) से एक अनुदान द्वारा उपलब्ध कराई गई निधियों द्वारा समर्थित है.

1. लंदन स्कूल आफ इकोनॉमिक्स तैयारी <ol><li> महिला मेंढ़कों (Xenopus laevis) अंडा संग्रह के लिए दो बार इंजेक्शन हैं. 1 इंजेक्शन (भड़काना) मेंढक प्रति 100 यू PMSG (गर्भवती घोड़ी सीरम Gonadotropin) है. मेंढक कम से कम दो दिनों primed किया ज...

<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>

डीएनए की क्षति Xenopus अंडा अर्क का उपयोग कर जांच की चौकी के अध्ययन में कई फायदे हैं. अंडे के अर्क का उपयोग सेल से मुक्त कोशिका चक्र के अंतरावस्था पर सिंक्रनाइज़ निष्कर्षों की एक बड़ी मात्रा प्रदान करता ?...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> अभिकर्मकों </td></tr><tr><td> विरोधी Chk1 एंटीबॉडी P-S344 </td><td> सेल संकेतन </td><td> 2348L </td><td></td></tr><tr><td> विरोधी Chk1 एंटीबॉडी </td><td> सांता क्रुज़ </td><td> SC7898 </td><td></td></tr><tr><td> Aprotinin </td><td> सांसद BIOMEDICALS </td><td> 0219115880 </td><td></td></tr><tr><td> Cycloheximide </td><td> सिग्मा </td><td> C7698-5G </td><td></td></tr><tr><td> Cytochalasin बी </td><td> ईएमडी </td><td> 250233 </td><td></td></tr><tr><td> Dithiothreitol (डीटीटी) </td><td> VWR </td><td> JTF780-2 </td><td></td></tr><tr><td> एचसीजी </td><td> सिग्मा </td><td> CG10 - 10VL </td><td></td></tr><tr><td> एल Cysteine </td><td> सिग्मा </td><td> C7352-1KG </td><td></td></tr><tr><td> Leupeptin </td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td> मिथाइल methanesulfonate (एमएमएस) </td><td> सिग्मा </td><td> 129,925 - 5G </td><td></td></tr><tr><td> Nocodazole </td><td> सिग्मा </td><td> M1404 - 2mg </td><td></td></tr><tr><td> PMSG </td><td> Calbiochem </td><td> 367222 </td><td></td></tr><tr><td> नमूना बफर </td><td> सिग्मा </td><td> S3401 </td><td></td></tr><tr><td> Tautomycin </td><td> Wako रसायन यूएसए </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> उपकरण </td></tr><tr><td> अंडे बिछाने के लिए बाल्टी </td><td> Rubbermaid वाणिज्यिक उत्पादों </td><td> 6308 </td><td></td></tr><tr><td> झूलते बाल्टी रोटर साथ CL2 आईईसी अपकेंद्रित्र </td><td> थर्मो वैज्ञानिक </td><td> 004260F </td><td></td></tr><tr><td> HB6 झूलते बाल्टी रोटर </td><td> थर्मो वैज्ञानिक </td><td> 11860 </td><td></td></tr><tr><td> Sorvall RC6 प्लस superspeed अपकेंद्रित्र </td><td> थर्मो वैज्ञानिक </td><td> 46910 </td><td></td></tr><tr><td> यूवी crosslinker </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> समाधान </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 मिमी NaCl, 2 मिमी KCl, 0.5 मिमी MgSO 4, 2.5 मिमी 2 CACL, 5 मिमी HEPES, 10 एम NaOH के साथ 7.8 पीएच को समायोजित </td></tr><tr><td> Aprotinin / Leupeptin स्टॉक </td><td></td><td></td><td> 10 मिलीग्राम / प्रत्येक पानी में मिलीलीटर. -80 डिग्री सेल्सियस पर 20 μl aliquots स्टोर </td></tr><tr><td> बफर एक्स </td><td></td><td></td><td> 0.2 एम sucrose, 80 मिमी KCl, 15 मिमी NaCl, 5 मिमी MgCl 2, 1 मिमी EDTA, 10 मिमी HEPES, एचसीएल द्वारा 7.5 पीएच को समायोजित </td></tr><tr><td> Cycloheximide स्टॉक </td><td></td><td></td><td> 10 मिलीग्राम / मिलीलीटर पानी में. स्टोर 1 -20 डिग्री सेल्सियस पर मिलीलीटर aliquots </td></tr><tr><td> Cytochalasin बी शेयर </td><td></td><td></td><td> 5 मिलीग्राम / मिलीलीटर DMSO में. -20 डिग्री सेल्सियस पर 20 μl aliquots स्टोर </td></tr><tr><td> Dithiothreitol स्टॉक (डीटीटी) </td><td></td><td></td><td> पानी में 1 एम. स्टोर 1 -20 डिग्री सेल्सियस पर मिलीलीटर aliquots </td></tr><tr><td> ईएलबी </td><td></td><td></td><td> 0.25 एम sucrose, 1 मिमी डीटीटी, 50 ग्राम / मिलीलीटर cycloheximide, 2.5 मिमी 2 MgCl, 50 मिमी KCl, 10 मिमी HEPES, pH7.7 </td></tr><tr><td> Nocodazole स्टॉक </td><td></td><td></td><td> 10 मिलीग्राम / मिलीलीटर DMSO में. -80 डिग्री सेल्सियस पर 5 μl aliquots स्टोर </td></tr><tr><td> ऊर्जा मिश्रण </td><td></td><td></td><td> 375 मिमी creatine फॉस्फेट, 50 मिमी, एटीपी, और 25 मिमी 2 MgCl. Aliquots -80 डिग्री सेल्सियस पर बच रहे हैं </td></tr><tr><td> परमाणु डाई समाधान </td><td></td><td></td><td> 0.4 छ / मिलीलीटर Hoechst 33258, 25% ग्लिसरॉल(V / v), में 1x पीबीएस </td></tr><tr><td> Tautomycin स्टॉक </td><td></td><td></td><td> DMSO में 100 सुक्ष्ममापी. -80 डिग्री सेल्सियस पर 10 μl aliquots स्टोर </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 अंडा निकालने के लिए डीएनए की क्षति जांच की चौकी की जांच के लिए एक उपयोगी मॉडल प्रणाली है. इस प्रोटोकॉल Xenopus अंडा अर्क और डीएनए की क्षति जांच की चौकी उत्प्रेरण अभिकर्मकों की तैयारी के लिए है. इन तकनीकों में डीएनए की क्षति जांच की चौकी संकेतन के अध्ययन में डीएनए हानिकारक दृष्टिकोण की एक किस्म के लिए अनुकूलनीय हैं.

डीएनए की क्षति का उपयोग चेकप्वाइंट के अध्ययन<em&gt; Xenopus</em&gt; Egg अर्क

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

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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