Name: steady-state, pre-steady-state, and single-turnover kinetic measurement for dna glycosylase activity
Uploaded: 2013-08-19T12:00:00
Description: Watch this Scientific Journal Video about Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity at JoVE.com

我们感谢朱莉·霍顿博士批判性阅读的手稿和拉金德拉·普拉萨德博士有用的建议和讨论。这项研究最初发表在杂志“生物化学佐佐A 等部分。人，“DNA序列背景效应的糖基化酶活性的人8 -羟基鸟嘌呤DNA糖基化酶。”J Biol Chem的 287，36702-36710（2012）2。这项工作是支持的全部或部分，由美国国立卫生研究院资助计划Z01-ES050158，NIEHS院内研究计划。

1。酶和DNA底物的制备<ol><li>过作为GST融合蛋白在大肠杆菌中的表达OGG1 大肠杆菌 ，利用GST标签进行净化，然后取出GST标签裂解HRV-3C蛋白酶（ 图1）6。 </li><li>购买化学合成的5&#39;-6-羧基（6-FAM）标记的寡核苷酸包含一个8 oxoG残留和互补未标记的寡核苷酸链。的34聚体寡核苷酸的5&#39;-末端的17位中包含一个8-oxoG。这些寡核苷酸，通过聚丙烯酰胺凝胶电泳纯化。 </li><...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+versus+repair:+NEIL1+removal+of+hydantoin+lesions+in+single-stranded,+bulge,+bubble,+and+duplex+DNA+contexts.">Mutation versus repair: NEIL1 removal of hydantoin lesions in single-stranded, bulge, bubble, and duplex DNA contexts.</a> Biochemistry. 49, 1658-1666 (2010).</li><li>Leipold, M. D., Workman, H., Muller, J. G., Burrows, C. J., David, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+and+removal+of+oxidized+guanines+in+duplex+DNA+by+the+base+excision+repair+enzymes+hOGG1,+yOGG1,+and+yOGG2.">Recognition and removal of oxidized guanines in duplex DNA by the base excision repair enzymes hOGG1, yOGG1, and yOGG2.</a> Biochemistry. 42, 11373-11381 (2003).</li><li>Robey-Bond, S. M., Barrantes-Reynolds, R., Bond, J. P., Wallace, S. S., Bandaru, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+acetobutylicum+8-oxoguanine+DNA+glycosylase+(Ogg)+differs+from+eukaryotic+Oggs+with+respect+to+opposite+base+discrimination.">Clostridium acetobutylicum 8-oxoguanine DNA glycosylase (Ogg) differs from eukaryotic Oggs with respect to opposite base discrimination.</a> Biochemistry. 47, 7626-7636 (2008).</li><li>Jarem, D. A., Wilson, N. R., Delaney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-dependent+DNA+damage+and+repair+in+a+trinucleotide+repeat+sequence.">Structure-dependent DNA damage and repair in a trinucleotide repeat sequence.</a> Biochemistry. 48, 6655-6663 (2009).</li><li>Livingston, A. L., Kundu, S., Henderson Pozzi, M., Anderson, W. D., David, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+roles+of+tyrosine+82+and+glycine+253+in+the+Escherichia+coli+adenine+glycosylase+MutY.">Insight into the roles of tyrosine 82 and glycine 253 in the Escherichia coli adenine glycosylase MutY.</a> Biochemistry. 44, 14179-14190 (2005).</li><li>Kundu, S., Brinkmeyer, M. K., Livingston, A. L., David, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenine+removal+activity+and+bacterial+complementation+with+the+human+MutY+homologue+(MUTYH)+and+Y165C,+G382D,+P391L+and+Q324R+variants+associated+with+colorectal+cancer.">Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.</a> DNA Repair. 8, 1400-1410 (2009).</li><li>Zharkov, D. O., Rosenquist, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+mammalian+8-oxoguanine-DNA+glycosylase+by+cadmium(II):+implications+for+cadmium+genotoxicity.">Inactivation of mammalian 8-oxoguanine-DNA glycosylase by cadmium(II): implications for cadmium genotoxicity.</a> DNA Repair. 1, 661-670 (2002).</li><li>Jarem, D. A., Wilson, N. R., Schermerhorn, K. M., Delaney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+persistence+of+8-oxo-7,8-dihydroguanine+within+a+hairpin+intermediate+exacerbates+a+toxic+oxidation+cycle+associated+with+trinucleotide+repeat+expansion.">Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion.</a> DNA Repair. 10, 887-896 (2011).</li><li>Mokkapati, S. K., Wiederhold, L., Hazra, T. 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动力学这里描述的方法，概括的方法来定义基本的动力学常数。如果一个时间过程的产品形成了第一种酶营业额迅速发生双相，然后经化学的一个步骤是在随后的催化失误限速。 OGG1的情况下，可以使用高浓度的酶与限制（S &lt;E)或高(S&gt;É）DNA的浓度测量的第一个营业额。在第一种情况下，该反应是有限的“单一营业额，并提供一定程度的酶的活性位点的催化速率。在后者的情况下，被评估多种?...

一个有氧环境加快基因组不稳定。甲的主要promutagenic DNA病变导致的氧化应激是7,8 - 二氢-8 - 羟基鸟嘌呤（8-oxoG）。这是由于8-oxoG的暧昧编码潜力。 8 oxoG发起碱基切除修复负责人8 - 羟基鸟嘌呤DNA糖基化酶（OGG1）。 OGG1切除8 oxoG基地，导致一个的嘌呤网站（AP站点）产品DNA糖基化酶活性。在某些情况下，一个弱OGG1裂解酶活性可以切割的AP站点。 一般动力学特性的DNA糖基化酶发现，他们表现出双相时间课程。在经过最初的快相产物的形成（ 即脉冲串），观察到的线性稳态阶段1-3。这种行为是表示化学（ 即构象变化或产品释放）过程中的时间过程的线性部分的限速的步骤，而钻针圣相，通常被称为“过渡阶段，在第一个周期的反应产物的形成，可在酶的活性位点相对应。当产品发布速率限制在稳态阶段，活动的测量提供了定性测量产品DNA结合的亲和力，但没有提供有关的酶的活性部位（ 即化学）事件的动力学信息。因此，需要的方法来分离和测量指数的预稳态脉冲串相位探测事件的过程中的第一酶在酶的活性位点4的营业额。 有三种标准的动力学方法OGG1催化行为的特征，（1）稳定状态，（2）预稳定的状态，和（3）单成交。这些方法彼此不同的酶的浓度在反应混合物中的每一种方法中使用的酶底物比。在一个典型的稳态的方法，有时也被称为多周转动力学，使用低浓度的酶按照产物的形成。当底物浓度大大超过酶的浓度，以便多个酶失误不显着影响底物浓度。在这种情况下，时间场应该是线性的，它往往是难以辨别是否发生突发过程中的第一个营业额，由于在这种方法中使用的酶浓度低;注意，脉冲串的幅度是酶的浓度相当于。这是可以克服的，通过使用较高的酶浓度和外推线性时间当然零时间检测是否很快就发生了第一种酶营业额。纵坐标（y轴）上的截距应该是酶的浓度成比例，并提供了一​​个衡量积极从事与底物的酶。虽然这种方法原则上可以提供证据的存在突发阶段，DIFferent方法需要测量的动力学突发阶段。在许多情况下，的突发阶段是太快了人工搅拌淬火技​​术测量。在这种情况下，预稳态和单营业额动能（ 即瞬态动能）的方法往往需要快速混合和猝灭反应5按照早的时间点的仪器。在一个稳定状态前的方法，使用高浓度的酶，以便在第一个营业额，形成显着数量的产品。 ，由于多个失误其次观察催化循环（ 即突发如下的线性相位），底物浓度是大于酶浓度（[酶] &lt;[基板]）。隔离事件的酶的活性部位无催化单车，单营业额的条件下被使用。在这种情况下，底物是饱和的酶（E &gt;&gt; S），以使所有的基片将参加我在单笔成交金额，通常会表现出一个单一的指数的时间过程。 如上所述酶表现出一个突发阶段，产品发布（K掉 ）往往限制的稳态阶段的时间过程的速率。产品放行的速度（V SS，浓/时间）可确定从斜坡的线性稳态阶段。活性酶浓度（E）产品发布的速度是需要转换的内在速率常数K掉 = V SS / [E]。重要的是，活性酶的浓度一般低于所测得的蛋白浓度因杂质，处于非活动状态的酶，酶的非生产性基板结合，和所使用的方法，以确定蛋白浓度。活性酶的浓度可以由突发幅度当产品释放缓慢的。因此，推断一个稳定状态的时间当然要零时间提供了一个估计需要从观察到的稳态率计算K掉 （产品发布）的活性酶。 要测量的动力学的突发，预稳态的方法是在第一营业额前发生的线性稳态阶段，要遵循产品的形成。该突发动力学如下酶产品的中间体的形成。一旦反应过程是由酶与底物混合的酶产品的量迅速增加，直至反应达到稳定状态阶段。如果催化比产品的发布更迅速，脉冲串的幅度是等于积极参与的酶和所观察到的指数接近平衡（十一观测值 ）对应的底物向产物的化学转化率，假设反向速率化学是可以忽略不计。 在某些情况下催化CY保鲜干扰预稳态分析，如化学和产品发布率的幅度并不显着不同。在这种情况下，采用酶相对过剩防止绑定到一个单一的营业额酶的催化循环和限制基板基板。因此，第一化学反应步骤可以分离和精确地确定作为一级反应速率常数（k 观测值 ）。此速率常数为k应该是相似的OBS从上述预稳态的方法确定。 这里，我们描述如何将这些动力学的方法，可以用来分析OGG1糖基化酶活性的。

<table border="1">
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 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
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 <td>5'-6-FAM labeled oligonucleotides containing a single 8-oxoG</td>
 <td>Eurofins MWG Operon</td>
 <td></td>
 <td>5'-P32 radiolabeled oligonucleotides can be used as well. Polyacrylamide gel purification grade is recommended. </td>
 </tr>
 <tr>
 <td>Unlabeled oligonucleotides (complementary strand)</td>
 <td>Eurofins MWG Operon</td>
 <td></td>
 <td>Polyacrylamide gel purification grade is recommended. </td>
 </tr>
 <tr>
 <td>1 ml BD Luer Lock disposable syringe</td>
 <td>BD Medical</td>
 <td>309628</td>
 <td>Lue Lock disposable syringe from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>10 ml BD Luer Lock disposable syringe</td>
 <td>BE Medical</td>
 <td>309604</td>
 <td>Luer Lock disposable syringe from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Circulating water bath</td>
 <td>Any vender</td>
 <td></td>
 <td></td>
 </tr>
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 <td>RQF-3 Rapid Quench-Flow Instrument</td>
 <td>KinTek Corporation</td>
 <td></td>
 <td>Rapid Quench-Flow Instrument from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>Typhoon Phosphorimager 8600</td>
 <td>GE Healthcare Life Sciences</td>
 <td></td>
 <td>Imager from other vendors can be used as well.</td>
 </tr>
 <tr>
 <td>KaleidaGraph</td>
 <td>Synergy Software</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

steady-state, pre-steady-state, and single-turnover kinetic measurement for dna glycosylase activity

人类8 - 羟基鸟嘌呤DNA糖基化酶（OGG1），切除从DNA诱变氧化性DNA损伤-8 - 氧代-7,8 - dihydroguanine（8-oxoG）。 OGG1动力学特性进行测量8 oxoG切除率和产品发布。当的OGG1浓度的低于底物DNA，产物的形成的时间过程是双相的，随后由一个线性稳态阶段产品形成的快速指数阶段（ 即脉冲串）。产物的形成的初始脉冲串对应的正确啮合在基片上的酶的浓度，和脉冲串的幅度取决于酶的浓度。一阶速率常数的突发对应的内在8 oxoG切除率和速度较慢的稳态测量产品的发行率（产品DNA解离速率常数，k 关闭 ）。在这里，我们描述了稳定状态，稳定状态，单营业额的方法来隔离和测量SPecific步骤在OGG1催化的循环。含病变的荧光标记的寡核苷酸和纯化OGG1是用来方便精确的动力学测量。由于酶浓度低，用来做稳态测量，人工搅拌反应试剂和淬火，可以进行确定的稳态率（K 关闭 ）。此外，外推在零时间，纵轴上的点的稳态速率表明的产物的形成过程中发生一个脉冲串的第一个营业额（ 即 y轴截距为正）。可以测量使用的是快速混合和猝灭技术，检查形成在短的时间间隔（&lt;1秒）之​​前的稳态阶段的产品的量和对应的速率为8的一级速率常数的指数的脉冲串相位oxoG切除术（化学）。化学步骤也可以使用一个单一的营业额的方法测量催化循环防止饱和底物DNA酶（E&gt; S）。这些方法可以测量的基本速率常数的影响的效率去除的DNA损伤。

稳态动力学分析是由使 ​​用200纳米的DNA底物和四种不同浓度明显OGG1（15，30，45和60纳米）确定通过Bradford蛋白检测2。时间课程适合产品形成一个线性方程来确定y轴截距，分别为2.2，11，15，和26纳米，分别相对于每个蛋白浓度（ 图2B）。的y截距，进一步绘制相对于每个实际的蛋白质浓度（ 图2C）。从图2C中的线的斜率，被确定为38％的活性酶的馏分?...

Watch this Scientific Journal Video about Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity at JoVE.com

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

时间8 - 羟基鸟嘌呤DNA糖苷酶糖基化酶活性课程双相参展产品的形成和爆发出的线性稳态阶段。利用淬火流技术中，突发和可以测量的稳态速率，分别对应到8 - 羟基鸟嘌呤和释放从产物DNA糖基化酶切除。

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Research

JoVE Journal

Chemistry

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

该DNA氧化标志物的高效液相色谱法测量，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷，在培养细胞和动物组织

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

科研

化学

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

对于DNA折纸分析和实验准备云母和硅衬底

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

乙烯聚合中使用并联电抗器的压力和链转移聚合的动力学分析

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

Fabrication and Testing of Photonic Thermometers

光子温度计的制作与测试

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

基于NMR的活性测定,用于测定核苷裂解酶的化合物抑制、IC50值、伪影活性和全细胞活性

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...