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

Эта работа частично поддержана средств, предоставленных Университетом Северной Каролины в Шарлотте, Wachovia основу фонда для профессорско-преподавательского мастерства, и грант от NIGMS (R15GM101571).

1. Подготовка LSE <ol><li> Женский лягушек (Xenopus Хепориз) вводится два раза для сбора яиц. Первая инъекция (грунтовка) составляет 100 U PMSG (Беременные Mare сыворотке гонадотропина) в лягушку. Лягушки должны быть загрунтованы по крайней мере, за два дня до вызывающие откладки яиц и загрунтоват?...

<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>

Есть несколько преимуществ в изучении контрольно-пропускного пункта повреждения ДНК использовании Xenopus яйцо экстрактов. Использование яйцо экстракт содержит большое количество бесклеточных экстрактов синхронизированы в интерфазе клеточного цикла. Яйцо экстракты могут быть ле...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Реагенты </td></tr><tr><td> Anti-Chk1 P-S344 антитела </td><td> Cell Signaling </td><td> 2348L </td><td></td></tr><tr><td> Anti-Chk1 антител </td><td> Санта-Крус </td><td> SC7898 </td><td></td></tr><tr><td> Апротинин </td><td> MP Biomedicals </td><td> 0219115880 </td><td></td></tr><tr><td> Циклогексимид </td><td> Сигма </td><td> C7698-5G </td><td></td></tr><tr><td> Цитохалазин </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> ХГЧ </td><td> Сигма </td><td> CG10-10VL </td><td></td></tr><tr><td> L-цистеин </td><td> Сигма </td><td> C7352-1кг </td><td></td></tr><tr><td> Лейпептин </td><td> VWR </td><td>97063-922 </td><td></td></tr><tr><td> Метиловый метансульфонат (MMS) </td><td> Сигма </td><td> 129925-5G </td><td></td></tr><tr><td> Нокодазолом </td><td> Сигма </td><td> M1404-2MG </td><td></td></tr><tr><td> ГСЖК </td><td> Calbiochem </td><td> 367222 </td><td></td></tr><tr><td> Пример буфер </td><td> Сигма </td><td> S3401 </td><td></td></tr><tr><td> Tautomycin </td><td> Wako Chemicals США </td><td> 209-12041 </td><td></td></tr><tr><td></td><td></td><td></td><td> Оборудование </td></tr><tr><td> Ковш для откладки яиц </td><td> Rubbermaid Commercial Products </td><td> 6308 </td><td></td></tr><tr><td> CL2 IEC центрифуге с бакет ротора </td><td> Thermo Scientific </td><td> 004260F </td><td></td></tr><tr><td> HB6 качающийся ротор ведро </td><td> Thermo Scientific </td><td> 11860 </td><td></td></tr><tr><td> Sorvall RC6 плюс сверхскоростные центрифуги </td><td> Thermo Scientific </td><td> 46910 </td><td></td></tr><tr><td> УФ сшивателя </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Решения </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 мМ NaCl, 2 мМ KCl, 0,5 мМ MgSO 4, 2,5 мМ CaCl 2, 5 мМ HEPES, рН до 7,8 с помощью 10 М NaOH </td></tr><tr><td> Апротинин / лейпептин складе </td><td></td><td></td><td> 10 мг / мл в воде. Храните 20 мкл аликвоты при -80 ° C. </td></tr><tr><td> Буфер X </td><td></td><td></td><td> 0,2 М сахарозы, 80 мМ KCl, 15 мМ NaCl, 5 мМ MgCl 2, 1 мМ ЭДТА, 10 мМ HEPES, отрегулировать рН до 7,5 соляной кислотой </td></tr><tr><td> Циклогексимид складе </td><td></td><td></td><td> 10 мг / мл в воде. Магазин 1 мл аликвоты при -20 ° C. </td></tr><tr><td> Цитохалазин складе </td><td></td><td></td><td> 5 мг / мл в ДМСО. Храните 20 мкл аликвоты при -20 ° C. </td></tr><tr><td> Dithiothreitol (DTT) акции </td><td></td><td></td><td> 1 M в воде. Магазин 1 мл аликвоты при -20 ° C. </td></tr><tr><td> ELB </td><td></td><td></td><td> 0,25 М сахарозы, 1 мМ DTT, 50 мкг / мл циклогексимид, 2,5 мМ MgCl 2, 50 мМ KCl, 10 мМ HEPES, pH7.7 </td></tr><tr><td> Нокодазолом складе </td><td></td><td></td><td> 10 мг / мл в ДМСО. Храните 5 мкл аликвоты при -80 ° C. </td></tr><tr><td> Энергия смеси </td><td></td><td></td><td> 375 мм креатин фосфат, 50 мМ ATP и 25 мМ MgCl 2. Аликвоты сохраняются при температуре -80 ° C. </td></tr><tr><td> Ядерная раствор красителя </td><td></td><td></td><td> 0,4 мкг / мл Hoechst 33258, 25% глицерина(V / V), в 1x PBS </td></tr><tr><td> Tautomycin складе </td><td></td><td></td><td> 100 мкм в ДМСО. Храните 10 мкл аликвоты при -80 ° 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 яйцо экстракт является полезной модели системы для расследования контрольно-пропускного пункта повреждения ДНК. Этот протокол предназначен для подготовки Xenopus яйцо экстракты и повреждение ДНК реагенты контрольно-пропускном пункте вызывающим. Эти методы могут быть адаптированы к различным повреждающим ДНК подходы в изучении контрольно-пропускного пункта сигнализации повреждения ДНК.

Исследование Checkpoint повреждения ДНК использовании<em&gt; Xenopus</em&gt; Экстракты Egg

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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