Name: kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins
Uploaded: 2017-02-25T15:00:00
Description: Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

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.).

NOTA: Siga todas as normas institucionais relativas à utilização segura e eliminação de material radioactivo, incluindo, mas não limitado a, o uso de equipamento de protecção individual, como luvas, óculos de segurança, jaleco, e escudos de acrílico adequados. NOTA: O tampão padrão consiste em 40 mM HEPES-KOH, pH 7,5, glutamato de potássio 50 mM, DTT 5 mM, EDTA 0,1 mM (tampão esta é pré-fabricados, a uma concentração 5x) e 0,3 mM de cada dNTP. Proteínas de replicação de...

<ol>
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A single-stranded DNA-binding protein.</a> J Biol Chem. 267 (21), 15022-15031 (1992).</li><li>Frick, D. N., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+primases.">DNA primases.</a> Annu Rev Biochem. 70, 39-80 (2001).</li><li>Lee, S. J., Richardson, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+lysine+residues+in+the+RNA+polymerase+domain+of+the+gene+4+primase-helicase+of+bacteriophage+T7.">Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.</a> J Biol Chem. 276 (52), 49419-49426 (2001).</li><li>Cao, W., De La Cruz, E. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fitting+enzyme+kinetic+data+with+KinTek+Global+Kinetic+Explorer.">Fitting enzyme kinetic data with KinTek Global Kinetic Explorer.</a> Methods Enzymol. 467, 601-626 (2009).</li><li>Johnson, 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+century+of+enzyme+kinetic+analysis,+1913.">A century of enzyme kinetic analysis, 1913.</a> FEBS Lett. 587 (17), 2753-2766 (2013).</li><li>Biswas, T., Resto-Roldan, E., Sawyer, S. K., Artsimovitch, I., Tsodikov, O. 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O factor mais importante na realização destas experiências é a disponibilidade da enzima purificada altamente activa. Durante o nosso trabalho com GP4, por exemplo, verificou-se que o armazenamento da enzima purificada em tampão (fosfato de potássio 20 mM, pH 7,4, EDTA 0,1 mM, DTT 1 mM) contendo 50% de glicerol a -20 ° C, leva a uma redução a actividade específica da preparação ao longo de alguns meses. Portanto, agora flash-freeze pequenas alíquotas de GP4 purificada em Tris-HCl a 25, pH 7,5, NaCl 50 mM, E...

A replicação de ADN é uma característica conservada de toda a vida celular. Enquanto a identidade e número de componentes de replicação variam amplamente, os mecanismos gerais são compartilhadas entre a evolução 1. Aqui, descrevemos, experimentos cinéticos descontínuos à base de gel, utilizando substratos radioativos, visando a compreensão da cinética de síntese de DNA atraso vertente in vitro por componentes do replisome T7. A maquinaria de replicação do bacteriófago T7 é muito simples, consistindo em apenas quatro proteínas (a polimerase de ADN, GP5, e o seu factor de processabilidade, tioredoxina de E. coli, os GP4 primase-helicase bifuncionais, e a proteína de ligação ao ADN de cadeia simples, GP2. 5) 2. Esta característica torna-se um sistema de modelo atractivo para estudar os mecanismos bioquímicos conservadas envolvidas na replicação do ADN. Especial ênfase é colocada sobre a formação de primers ribonucleï¿½ido por DNA T7 primase, uma critical passo na iniciação de síntese de ADN. Além disso, pode-se também analisar a utilização desses iniciadores por ADN-polimerase de T7 ou outras proteínas de replicação através da sua inclusão na mistura de reacção. A primase de T7-helicase, GP4, catalisa a formação de iniciadores para a síntese de DNA em sequências de ADN específicos chamados locais de reconhecimento, ou primase PRS, (5&#39;-TCG-3 &#39;). A citosina no PRS é enigmática, é essencial para o reconhecimento do local, mas não é copiado para o produto 3. O primeiro passo na síntese do iniciador por GP4 envolve a formação do dinucleótido pppAC, que é então estendida a um trímero, e, eventualmente, a iniciadores tetranucleotídeo funcionais pppACCC, pppACCA, ou pppACAC dependendo da sequência no molde 4. Estes iniciadores podem então ser utilizados por ADN-polimerase de T7 para iniciar a síntese de ADN, que é também um processo assistido por GP4 5, 6. A este respeito, o primasedomínio estabiliza os tetraribonucleotides extremamente curtos com o modelo, impedindo a sua dissociação, envolve polimerase ADN de uma forma conducente a garantir o primer / modelo no sítio ativo 7 polimerase. Estes passos (a síntese de ARN do iniciador, a polimerase de handoff iniciador, e extensão) são repetidos em ciclos múltiplos de replicar a costa de retardamento e tem de ser coordenado com a replicação da costa principal. Os ensaios descritos aqui são altamente sensíveis e podem ser executadas num período de tempo moderado. No entanto, eles são relativamente de baixo rendimento e um grande cuidado deve ser exercido no uso e descarte de materiais radioativos. Dependendo da velocidade à qual a reacção prossegue, pode-se empregar um instrumento de têmpera rápida para atingir as amostras em escalas de tempo favorável para análise significativa de cursos de tempo da reacção, quer em contínuo ou no estado pré-estável. Recentemente, usamos ensaios descritos aqui para fornecer evidence a importância da introdução do iniciador a partir do domínio de primase de GP4 na iniciação de fragmentos de Okazaki. Além disso, encontramos evidências de um papel regulador para a proteína, gp2.5 ligação do T7 DNA de cadeia simples, na promoção da formação de primer eficiente e utilização 8.

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			Tris pre-set crystals 
			pH 7.5</td>
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			<td height="42" style="height:42px;width:216px;">L-Glutamic acid potassium salt monohydrate</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">G1501&#160;</td>
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			<td height="63" style="height:63px;width:216px;">Magnesium chloride hexahydrate</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
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			<td height="21" style="height:21px;width:216px;">1,4-Dithiothreitol</td>
			<td style="width:83px;">J.T. Baker</td>
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			<td height="63" style="height:63px;width:216px;">Sodium Dodecyl Sulfate</td>
			<td style="width:83px;">MP Biomedicals</td>
			<td align="right" style="width:119px;">811030</td>
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			<td height="63" style="height:63px;width:216px;">(Ethylenedinitrilo) Tetraacetic acid, disodium salt, dihydrate</td>
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			<td height="42" style="height:42px;width:216px;">[&#945;-32P] CTP</td>
			<td style="width:83px;">PerkinElmer</td>
			<td style="width:119px;">BLU508H</td>
			<td>radioactive, take protective measures</td>
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			<td height="42" style="height:42px;width:216px;">[&#947;-32P] ATP</td>
			<td style="width:83px;">PerkinElmer</td>
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			<td>radioactive, take protective measures</td>
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			<td>four dNTPs part of set</td>
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			<td height="21" style="height:21px;width:216px;">100 mM ATP and CTP</td>
			<td style="width:83px;">Epicentre</td>
			<td style="width:119px;">RN02825</td>
			<td>part of NTP set</td>
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			<td height="84" style="height:84px;width:216px;">ssDNA constructs</td>
			<td style="width:83px;">Integrated DNA Technologies</td>
			<td style="width:119px;">N/A</td>
			<td>custom sequences&#160;</td>
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			<td height="63" style="height:63px;width:216px;">methanol&#160;</td>
			<td style="width:83px;">Macron Fine Chemicals</td>
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			<td height="42" style="height:42px;width:216px;">formamide</td>
			<td style="width:83px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">17899</td>
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			<td height="21" style="height:21px;width:216px;">bromophenol blue</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">B8026&#160;</td>
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			<td height="21" style="height:21px;width:216px;">cylene xyanol&#160;</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">X4126&#160;</td>
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			<td height="21" style="height:21px;width:216px;">acrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">A9099</td>
			<td>Toxic</td>
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			<td height="21" style="height:21px;width:216px;">N,N&#8242;-Methylenebisacrylamide</td>
			<td style="width:83px;">SIGMA</td>
			<td style="width:119px;">M7279&#160;</td>
			<td>Toxic</td>
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			<td height="63" style="height:63px;width:216px;">Urea</td>
			<td style="width:83px;">Boston Bioproducts</td>
			<td style="width:119px;">P-940</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Boric acid</td>
			<td style="width:83px;">Mallinckrodt Chemicals</td>
			<td align="right" style="width:119px;">2549</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rapid Quench-Flow Instrument&#160;</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">RQF-3&#160;</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kintek Explorer</td>
			<td style="width:83px;">Kintek Corp.</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kaleidagraph&#160;</td>
			<td style="width:83px;">Synergy Software</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
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kinetics of lagging-strand dna synthesis in vitro by the bacteriophage t7 replication proteins

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.

Os resultados mostrados na Figura 1A foram obtidos como descrito no passo 1 do protocolo, isto é, por amostragem manualmente uma reacção de síntese de primer catalisada por GP4 sob condições de turnover múltiplo. Aqui, uma gama de produtos, após electroforese em gel pode ser observado usando CTP marcado com 32 P na posição-α em reacções de síntese do iniciador (Figura 1A). O precursor marcado, CTP, mostr...

Watch this Scientific Journal Video about Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins at JoVE.com

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

Cinética da Síntese da Fita Atrasada de DNA  In Vitro pelas Proteínas de Replicação do Bacteriófago T7

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

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