Name: iterative optimization of dna duplexes for crystallization of seqa-dna complexes
Uploaded: 2012-11-01T15:00:00
Description: Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Forfatterne vil gerne takke PXRR personale på NSLS (Brookhaven National Laboratory) for assistance under dataindsamlingen og Monica Pillon for at få hjælp med DNA oprensning. Dette arbejde blev støttet af den canadiske Institutes of Health Research (MOP 67.189).

1. Proteinoprensning Den fleksible linker forbinder N-(oligomerisering) og C-terminal (DNA binding) domæner af SeqA hjælpemidler anerkendelsen af ​​hemimethyleret GATC repeats er adskilt af 1-3 omdrejninger på DNA&#39;et. Til denne undersøgelse anvendte vi et dimert variant af SeqA (SeqAΔ (41-59)-A25R) med en punktmutation i det N-terminale domæne, der forhindrer yderligere oligomerisering og en forkortet linker, som begrænser DNA binding til tandem GATC gentager adskilt udelukkende...

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En af de største udfordringer i makromolekylære røntgenkrystallografi er at få diffraktion kvalitet krystaller. I tilfælde af protein-eller protein-DNA-komplekser, er denne udfordring forværres på grund af de yderligere variabler, der skal optimeres. Det er en udbredt opfattelse, at længden af ​​DNA&#39;et og tilstedeværelsen af ​​klæbrige overhangs at øge associeringen af ​​tilstødende DNA-molekyler i en længere pseudo-duplex er de vigtigste parametre, der skal optimeres. Vi har imidlertid vist...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog # </td><td> Kommentarer (valgfrit) </td></tr><tr><td> TRIS </td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td> Ethylendiamintetraeddikesyre (EDTA) </td><td> Fisher Scientific </td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrol (DTT) </td><td> Bio Basic Inc. </td><td> DB0058 </td><td></td></tr><tr><td> NaCl </td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td> Glycerol </td><td> Caledon </td><td> 5350-1 </td><td></td></tr><tr><td> Saccharose </td><td> Sigma-Aldrich </td><td> S5016-500G </td><td></td></tr><tr><td> Natriumdodecylsulfat (SDS) </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td> Urea </td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40% 29:1 Bis / acrylamid </td><td> Bio Basic Inc. </td><td> A0007-500ml </td><td> Opbevares ved 4 ° C </td></tr><tr><td> Borsyre </td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td> Xylencyanol FF </td><td> Bio-Rad </td><td> 161-0423 </td><td></td></tr><tr><td> Bromphenol blå </td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td> Dual indstillelig Vertikal gelsystem </td><td> CBC Scientific Company Inc. </td><td> DASG-250 </td><td></td></tr><tr><td> Index krystallisering skærm </td><td> Hampton Research </td><td> HR2-144 </td><td> Opbevares ved 4 ° C </td></tr><tr><td> Wizard I krystallisering skærm </td><td> Emerald BioSystems </td><td> EBS-WIZ-1 </td><td> Opbevares ved 4 ° C </td></tr><tr><td> Wizard II krystallisering skærm </td><td&gt; Emerald BioSystems </td><td> EBS-WIZ-2 </td><td> Opbevares ved 4 ° C </td></tr><tr><td> Classics krystallisering skærm </td><td> Qiagen </td><td> 130.701 </td><td> Opbevares ved 4 ° C </td></tr><tr><td> Intelliplate bakker </td><td> Art Robbins Instruments </td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td> Løsninger Proteinoprensning buffer: 100 mM TRIS pH 8, 2 mM EDTA, 2 mM DTT og 5% glycerol. Protein storage buffer: 20 mM TRIS pH 8, 150 mM NaCI, 5 mM DTT, 0,5 mM EDTA og 5% glycerol. Gel loading mix: Tilsæt 20 g saccharose, 25 mg bromphenolblåt, 25 mg xylencyanol FF, 1 ml 10% vægt / vol SDS og 10 ml 10X TBE til 70 ml autoklaveret ddH 2 O. Der omrøres under mild opvarmning indtil saccharose er opløst, og justere den endeligevolumen til 100 ml med autoklaveret ddH 2 O. Opbevares ved 4 ° C. 2X loading buffer: Tilføj 11 g urinstof og 10 ml gel loading mix. Der omrøres på en varmeplade, indtil urinstof opløses. Alikvot i 2 ml rør og opbevares ved 4 ° C. 10X PAGE mix: Bland 420,4 g urinstof, 100 ml 10X TBE (autoklaveret), 250 ml 40% 29:1 Bis / Acrylamid i ddH 2 O. Rør rundt indtil helt opløst og justere lydstyrken til 1 liter. Opbevares i mørke flasker ved 4 ° C. 10X TBE: Opløs 108 g af TRIS, 55 g borsyre og 9,3 g EDTA i 1 liter ddH 2 O. Autoklave og opbevares ved stuetemperatur. Elueringspuffer: Fortynd 8 ml af 5 M NaCl, 2 ml 1 M Tris pH 7,5, 0,4 ml 0,5 M EDTA pH 8 til 200 ml ddH 2 O. Autoklave og opbevares ved stuetemperatur. </td></tr></tbody></table&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

Escherichia coli SeqA er en negativ regulator af DNA-replikation, der forhindrer for tidlig reinitieringsaktivitet hændelser ved sekvestrering hemimethyleret GATC klynger i replikationsorigin 1. Ud over oprindelse, SeqA findes på replikationsgafler, hvor det organiserer nyligt replikeret DNA i højere ordnede strukturer 2. SeqA associerede kun svagt med enkelte GATC sekvenser, men danner høj affinitet komplekser med DNA-duplekser indeholder multiple GATC sites. Den minimale funktionel og strukturel enhed af SeqA er en dimer, hvilket forklarer kravet om mindst to GATC-sekvenser til dannelse af en høj-affinitet kompleks med hemimethyleret DNA 3. Derudover SeqA arkitektur med oligomerisering og DNA-bindende domæner adskilt af en fleksibel linker, tillader binding til GATC repeats er adskilt af op til tre spiralformede vindinger. Derfor forstå funktionen af ​​SeqA på et molekylært niveau kræver strukturelle analysis af SeqA bundet til flere GATC-sekvenser. I protein-DNA krystallisation, kan DNA har ingen til en ekstraordinær effekt på emballagen interaktioner afhængig af de relative størrelser og arkitektur af proteinet og DNA. Hvis proteinet er større end DNA eller fodspor fleste af DNA&#39;et, er krystallen emballagen primært medieret af protein-protein interaktioner. Omvendt, når proteinet er af samme størrelse eller mindre end DNA eller det kun omfatter en del af DNA, DNA-DNA og DNA-protein-interaktioner dominerer krystal pakning. Derfor krystallisation af protein-DNA-komplekser kræver systematisk screening af DNA længde 4 og DNA-ender (stump eller overhæng) 5-7. I denne rapport beskriver vi, hvordan man kan designe, optimere, rense og krystallisere hemimethyleret DNA-duplekser indeholdende tandem GATC repeats i kompleks med en dimer variant af SeqA (SeqAΔ (41-59)-A25R) til opnåelse af krystaller er egnede til strukturbestemmelse.

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Krystalstrukturen af ​​protein-DNA-komplekser kan give indsigt i proteinfunktion, mekanisme, såvel som af arten af ​​den specifikke interaktion. Her rapporterer vi hvordan man kan optimere længde, sekvens og ender duplex DNA for co-krystallisering med<em&gt; Escherichia coli</em&gt; SeqA, en negativ regulator af replikation indvielse.

Iterative Optimering af DNA-duplekser for Krystallisation af SeqA-DNA-komplekser

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

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