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

Dette arbeidet støttes delvis av midler fra The University of North Carolina i Charlotte, Wachovia grunnlaget fond for fakultetet excellence, og en bevilgning fra NIGMS (R15GM101571).

1. LSE Forberedelse <ol><li> Kvinnelige frosker (Xenopus laevis) er injisert to ganger for egg samling. Den første injeksjonen (priming) er 100 U PMSG (gravid Mare Serum gonadotropin) per frosk. Frosker må primes minst to dager før inducing egglegging og primet froskene er holdbare i opptil to uker. Å prime frosker, injisere PMSG subkutant i rygg lymph sacs med en 3 ml sprøyte og 27 G nål. </li><li> Å indusere egglegging, injisere 500 U hCG (humant koriongonadotropin) per primet frosk subkutant i rygg ...

<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>

Det er flere fordeler ved å studere DNA skade sjekkpunkt med Xenopus egg ekstrakter. Bruken av egg ekstrakter gir en stor mengde celle-frie ekstrakter synkronisert ved interfase av cellesyklus. Egg ekstrakter kan enkelt og billig gjort. Det er relativt lett å skade DNA eller kromatin og for å avdekke en feil i DNA skade sjekkpunkt etter immunodepleting et mål protein fra egg ekstrakt. Deretter kan en potensiell funksjon defekt bli &quot;reddet&quot; av addback av vill type eller mutant rekombinante proteine...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagenser </td></tr><tr><td> Anti-Chk1 P-S344 antistoff </td><td> Cellesignalisering </td><td> 2348L </td><td></td></tr><tr><td> Anti-Chk1 antistoff </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> Ditiotreitol (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> Metylmetansulfonat (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> Prøvebuffer </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> Utstyr </td></tr><tr><td> Bøtte for å legge egg </td><td> Rubbermaid kommersielle produkter </td><td> 6308 </td><td></td></tr><tr><td> CL2 IEC sentrifuge med svingende spannrotor </td><td> Thermo Scientific </td><td> 004260F </td><td></td></tr><tr><td> HB6 svingende spannrotor </td><td> Thermo Scientific </td><td> 11860 </td><td></td></tr><tr><td> Sorvall RC6 pluss SuperSpeed ​​sentrifuge </td><td> Thermo Scientific </td><td> 46910 </td><td></td></tr><tr><td> UV tverrbinder </td><td> UVP </td><td> 95-0174-01 </td><td></td></tr><tr><td></td><td></td><td></td><td> Løsninger </td></tr><tr><td> 1x MMR </td><td></td><td></td><td> 100 mM NaCl, 2 mM KCl, 0,5 mM MgSO 4, 2,5 mM CaCl 2, 5 mM HEPES, justere pH til 7,8 med 10 M NaOH </td></tr><tr><td> Aprotinin / Leupeptin lager </td><td></td><td></td><td> 10 mg / ml hver i vann. Lagre 20 ul aliquoter ved -80 ° C. </td></tr><tr><td> Buffer X </td><td></td><td></td><td> 0,2 M sukrose, 80 mM KCl, 15 mM NaCl, 5 mM MgCl 2, 1 mM EDTA, 10 mM HEPES, justere pH til 7,5 ved HCl </td></tr><tr><td> Cycloheximide lager </td><td></td><td></td><td> 10 mg / ml i vann. Store 1 ml alikvoter ved -20 ° C. </td></tr><tr><td> Cytochalasin B lager </td><td></td><td></td><td> 5 mg / ml i DMSO. Lagre 20 ul aliquoter ved -20 ° C. </td></tr><tr><td> Ditiotreitol (DTT) lager </td><td></td><td></td><td> 1 M i vann. Store 1 ml alikvoter ved -20 ° C. </td></tr><tr><td> ELB </td><td></td><td></td><td> 0,25 M sukrose, 1 mM DTT, 50 ug / ml cycloheximid, 2,5 mM MgCl 2, 50 mM KCl, 10 mM HEPES, pH7.7 </td></tr><tr><td> Nocodazole lager </td><td></td><td></td><td> 10 mg / ml i DMSO. Lagre 5 pl aliquoter ved -80 ° C. </td></tr><tr><td> Energi Blanding </td><td></td><td></td><td> 375 mM kreatinfosfat, 50 mM ATP, og 25 mM MgCl 2. Aliquoter lagres ved -80 ° C. </td></tr><tr><td> Nuclear fargeløsning </td><td></td><td></td><td> 0,4 ug / ml Hoechst 33258, 25% glycerol(V / v), i 1x PBS </td></tr><tr><td> Tautomycin lager </td><td></td><td></td><td> 100 uM i DMSO. Lagre 10 pl aliquoter ved -80 ° C. </td></tr></tbody></table>

study of the dna damage checkpoint using xenopus egg extracts

På en daglig basis, blir cellene utsatt for en rekke av endogene og miljømessige fornærmelser. For å bekjempe disse fornærmelser, har cellene utviklet seg DNA-skader sjekkpunkt signaliserer som overvåking mekanisme for å oppfatte DNA skade og direkte cellulære responser til DNA-skader. Det er flere grupper av proteiner som kalles sensorer, givere og effektbokser involvert i DNA-skader sjekkpunkt signalering (figur 1). I dette komplekset signalveien, er ATR (ATM og Rad3-relatert) en av de store kinaser som kan svare på DNA skade og replikering stress. Aktivert ATR kan phosphorylate sine nedstrøms underlag som Chk1 (Checkpoint kinase 1). Følgelig fosforylert og aktiveres Chk1 fører til mange nedstrøms effekter i DNA skade sjekkpunkt inkludert cellesyklus, transkripsjon aktivering, DNA skade reparasjon, og apoptose eller senescence (figur 1). Når DNA er skadet, unnlater å aktivere DNA-skade sjekkpunkt resultater i unrepluftes skader og senere, genomisk ustabilitet. Studiet av DNA skade sjekkpunkt vil belyse hvordan cellene opprettholde genomisk integritet og gir en bedre forståelse av hvordan menneskelige sykdommer, så som kreft, utvikle. Xenopus laevis egg ekstrakter er fremstår som en kraftig celle-ekstrakt modellsystem i DNA-skader sjekkpunkt forskning. Lav hastighet ekstrakt (LSE) ble opprinnelig beskrevet av Masui gruppe 1. Tilsetningen av demembranated sperm kromatin til LSE resulterer i kjerner formasjonen der DNA er replikert i en semiconservative mote gang per cellesyklus. Den ATR/Chk1-mediated sjekkpunkt signalveien er utløst av DNA-skader eller replikering stress 2. To metoder brukes i dag til å indusere DNA skade sjekkpunkt: DNA skadelige tilnærminger og DNA skade-etterligne strukturer tre. DNA-skade kan induseres ved ultrafiolett (UV) stråling, γ-bestråling, metyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitrokinolin-1-oksyd (4-NQO), eller Aphidicolin 3, 4. MMS er et alkylerende agent som hemmer DNA replikasjon og aktiverer ATR/Chk1-mediated DNA skade sjekkpunkt 4-7. UV bestråling utløser også ATR/Chk1-dependent DNA skade sjekkpunkt 8. DNA skade-etterligne strukturen AT70 er en annealed kompleks av to oligonukleotider poly-(dA) 70 og poly-(dT) 70. Den AT70 systemet ble utviklet i Bill Dunphy laboratorium og er mye brukt for å indusere ATR/Chk1 checkpoint signalering 9-12. Her beskriver vi protokoller (1) for å forberede cellefri egg ekstrakter (LSE), (2) å behandle Xenopus sperm kromatin med to forskjellige DNA skade tilnærminger (MMS og UV), (3) for å forberede den DNA skade-etterligne strukturen AT70, og (4) for å utløse ATR/Chk1-mediated DNA skade sjekkpunkt i LSE med skadet sperm kromatin eller en DNA skade-etterligne struktur.

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 (Video) | JoVE

Xenopus egg ekstrakt er et nyttig modellsystem for å undersøke DNA-skader sjekkpunkt. Denne protokollen er for utarbeidelse av Xenopus egg ekstrakter og DNA-skade checkpoint inducing reagenser. Disse teknikkene kan tilpasses en rekke DNA skadelige tilnærminger i studiet av DNA skade sjekkpunkt signalering.

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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

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