Kinetik af efterslæb-DNA Synthesis<em> In vitro</em> Af bakteriofagen T7 replikationsproteiner
Summary
We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.
Abstract
Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.
Introduction
Replikationen af DNA er et konserveret egenskab ved alle cellulære liv. Mens identitet og antallet af replikation komponenter varierer meget, er de generelle mekanismer deles på tværs af evolution 1. Her beskriver vi gel-baseret, diskontinuerte kinetiske eksperimenter under anvendelse radioaktive substrater, der tager sigte på at forstå kinetikken af tilbagestående-DNA-syntese in vitro ved komponenter af T7 replisome. Replikation maskiner af bakteriofag T7 er meget enkel og består af kun fire proteiner (DNA-polymerasen, GP5, og dets processivitet faktor, E. coli thioredoxin, de bifunktionelle primase-helicase GP4, og det enkeltstrengede DNA-bindende protein, GP2. 5) 2. Denne funktion gør det til et attraktivt modelsystem til at studere de konserverede biokemiske mekanismer involveret i DNA-replikation.
Der lægges særlig vægt på dannelsen af ribonukleotid primere ved T7 DNA primase, en critical trin i initiering af DNA-syntese. Derudover kan man også undersøge brugen af disse primere ved hjælp af T7-DNA-polymerase eller andre replikationsproteiner ved at inkludere dem i reaktionsblandingen. T7-primase-helicase, GP4, katalyserer dannelsen af primere til DNA-syntese ved specifikke DNA-sekvenser kaldet primase genkendelsessteder eller PRS, (5'-GTC-3 '). Den cytosin i PRS er kryptisk, det er vigtigt for anerkendelse af hjemmesiden, men det er ikke kopieres ind i produkt 3. Det første trin i primer syntese ved GP4 involverer dannelsen af dinukleotidet pppAC, som derefter udvides til en trimer, og i sidste ende til funktionelle tetranukleotid primere pppACCC, pppACCA eller pppACAC afhængigt af sekvensen i skabelonen 4. Disse primere kan dernæst anvendes af T7-DNA-polymerase at initiere DNA-syntese, som også er et GP4-assisteret proces 5, 6. I denne forbindelse primasedomæne stabiliserer de ekstremt korte tetraribonucleotides med skabelonen, hindring af dissociering, indgriber DNA-polymerase på en måde bidrage til at sikre primer / skabelon i polymerase aktive sted 7. Disse trin (RNA primer syntese, primer handoff til polymerase, og udvidelse) er gentaget i flere cyklusser at kopiere halter streng og skal koordineres med replikation af de førende streng.
De her beskrevne assays er meget følsomme og kan udføres i en moderat tidsramme. Men de er relativt lav-throughput og stor omhu skal udvises i brug og bortskaffelse af radioaktive materialer. Afhængig af hastigheden, hvormed reaktionen forløber, kan man anvende et hurtigtvirkende quench instrument til at opnå prøver på tidsskalaer som kan underkastes en meningsfuld analyse af reaktionen tidsforløb i enten stabil eller præ-steady state. For nylig brugte vi assays beskrevet her for at give evidence af vigtigheden af primer frigivelse fra primase domæne GP4 i indledningen af Okazaki-fragmenter. Derudover fandt vi tegn på en regulerende rolle for T7 enkeltstrenget DNA-bindende protein, gp2.5, at fremme en effektiv primer dannelse og udnyttelse 8.
Protocol
Representative Results
Discussion
Den vigtigste faktor i at udføre disse eksperimenter er tilgængeligheden af højaktive oprenset enzym. Under vores arbejde med GP4, for eksempel fandt vi, at opbevaring af det oprensede enzym i buffer (20 mM kaliumphosphat, pH 7,4, 0,1 mM EDTA, 1 mM DTT) indeholdende 50% glycerol ved -20 ° C fører til en reduktion i den specifikke aktivitet af præparatet over et par måneder. Derfor har vi nu flash-freeze små portioner af oprenset GP4 i 25 mM Tris-HCI, pH 7,5, 50 mM NaCl, 0,1 mM EDTA, 1 mM TCEP, og 10% glyce…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
We thank C.C.R. laboratory members for comments and S. Moskowitz for figure preparation. This work was supported by National Institutes of Health Grants F32GM101761 (A.J.H.) and GM54397 (C.C.R.).
Materials
Tris pre-set crystals pH 7.5 |
SIGMA | T4128 | |
| Tris base | SIGMA | 93362 | |
| L-Glutamic acid potassium salt monohydrate | SIGMA | G1501 | |
| Magnesium chloride hexahydrate | Mallinckrodt Chemicals | 5958-04 | |
| 1,4-Dithiothreitol | J.T. Baker | F780-02 | |
| Sodium Dodecyl Sulfate | MP Biomedicals | 811030 | |
| (Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate | Mallinckrodt Chemicals | 4931-04 | |
| [α-32P] CTP | PerkinElmer | BLU508H | radioactive, take protective measures |
| [γ-32P] ATP | PerkinElmer | BLU502A | radioactive, take protective measures |
| 100 mM dATP, dCTP, dGTP, dTTP | Affymetrix | 77100 | four dNTPs part of set |
| 100 mM ATP and CTP | Epicentre | RN02825 | part of NTP set |
| ssDNA constructs | Integrated DNA Technologies | N/A | custom sequences |
| methanol | Macron Fine Chemicals | 3017-08 | |
| formamide | Thermo Scientific | 17899 | |
| bromophenol blue | SIGMA | B8026 | |
| cylene xyanol | SIGMA | X4126 | |
| acrylamide | SIGMA | A9099 | Toxic |
| N,N′-Methylenebisacrylamide | SIGMA | M7279 | Toxic |
| Urea | Boston Bioproducts | P-940 | |
| Boric acid | Mallinckrodt Chemicals | 2549 | |
| Rapid Quench-Flow Instrument | Kintek Corp. | RQF-3 | |
| Kintek Explorer | Kintek Corp. | N/A | |
| Kaleidagraph | Synergy Software | N/A |
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