Name: application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein
Uploaded: 2010-03-31T17:00:00
Description: Watch this Scientific Journal Video about Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein at JoVE.com

这项工作是由美国国家科学基金会职业奖（MMH），巴里M戈德华特奖学金（FNB）和ASBMB本科生研究奖（CWD）的支持。请提供由马丁博士韦伯（MRC，英国）的过度表达的PBP的克隆。

A.测量MSH2，MSH6的DNA结合动力学 1。 MSH2，MSH6 DNA结合动力学实验样品制备一个基于荧光动力学上停流的DNA结合实验试剂的制备是类似上荧光的平衡实验。事实上，平衡约束力的分析，应先估计的相互作用的解离常数（K D），以优化的动力学分析反应条件。停流实验需要平衡或稳态实验相比较大数量的生物材料，因此，这种方法是最可行的低蛋白...

<ol>
	<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=Advances+in+transient-state+kinetics.">Advances in transient-state kinetics.</a> Curr Opin Biotechnol. 9 (1), 87-89 (1998).</li><li>Johnson, K. A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Kinetic analysis of macromolecules. , (2003).</li><li>Obmolova, G., Ban, C., Hsieh, P., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+mismatch+repair+protein+MutS+and+its+complex+with+a+substrate+DNA.">Crystal structures of mismatch repair protein MutS and its complex with a substrate DNA.</a> Nature. 407 (6805), 703-710 (2000).</li><li>Lamers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+structure+of+DNA+mismatch+repair+protein+MutS+binding+to+a+G+x+T+mismatch.">The crystal structure of DNA mismatch repair protein MutS binding to a G x T mismatch.</a> Nature. 407 (6805), 711-717 (2000).</li><li>Warren, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+human+MutSalpha+DNA+lesion+recognition+complex.">Structure of the human MutSalpha DNA lesion recognition complex.</a> Mol Cell. 26 (4), 579-592 (2007).</li><li>Kunkel, T. A. &. a. m. p. ;. a. m. p., Erie, D. 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> DNA Mismatch Repair. Annu Rev Biochem. 74, 681-710 (2005).</li><li>Jiricny, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifaceted+mismatch-repair+system.">The multifaceted mismatch-repair system.</a> Nat Rev Mol Cell Biol. 7 (5), 335-346 (2006).</li><li>Hsieh, P., Yamane, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+mismatch+repair:+Molecular+mechanism,+cancer,+and+ageing.">DNA mismatch repair: Molecular mechanism, cancer, and ageing.</a> Mech Ageing Dev. 129 (7-8), 391-407 (2008).</li><li>Jacobs-Palmer, E., Hingorani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+nucleotides+on+MutS-DNA+binding+kinetics+clarify+the+role+of+MutS+ATPase+activity+in+mismatch+repair.">The effects of nucleotides on MutS-DNA binding kinetics clarify the role of MutS ATPase activity in mismatch repair.</a> J Mol Biol. 366 (4), 1087-1098 (2007).</li><li>Antony, E., Khubchandani, S., Chen, S., Hingorani, M. M., M, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+Msh2+and+Msh6+subunits+to+the+asymmetric+ATPase+and+DNA+mismatch+binding+activities+of+Saccharomyces+cerevisiae+Msh2-Msh6+mismatch+repair+protein.">Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2-Msh6 mismatch repair protein.</a> DNA Repair (Amst). 5 (2), 153-162 (2006).</li><li>Antony, E., Hingorani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mismatch+recognition-coupled+stabilization+of+Msh2-Msh6+in+an+ATP-bound+state+at+the+initiation+of+DNA+repair.">Mismatch recognition-coupled stabilization of Msh2-Msh6 in an ATP-bound state at the initiation of DNA repair.</a> Biochemistry. 42 (25), 7682-7693 (2003).</li><li>Finkelstein, J., Antony, E., Hingorani, M. M., O'Donnell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction+and+analysis+of+eukaryotic+multiprotein+complexes+in+Escherichia+coli+using+a+dual-vector+strategy.">Overproduction and analysis of eukaryotic multiprotein complexes in Escherichia coli using a dual-vector strategy.</a> Anal Biochem. 319 (1), 78-87 (2003).</li><li>Zhai, J., Hingorani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S.+cerevisiae+Msh2-Msh6+DNA+binding+kinetics+reveal+a+mechanism+of+targeting+sites+for+DNA+mismatch+repair.">S. cerevisiae Msh2-Msh6 DNA binding kinetics reveal a mechanism of targeting sites for DNA mismatch repair.</a> Proc Natl Acad Sci U S A. 107 (2), 680-685 (2010).</li><li>Brune, M., Hunter, J. L., Corrie, J. E., Webb, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct,+real-time+measurement+of+rapid+inorganic+phosphate+release+using+a+novel+fluorescent+probe+and+its+application+to+actomyosin+subfragment+1+ATPase.">Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase.</a> Biochemistry. 33 (27), 8262-8271 (1994).</li><li>Brune, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+inorganic+phosphate+interaction+with+phosphate+binding+protein+from+Escherichia+coli.">Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.</a> Biochemistry. 37 (29), 10370-10380 (1998).</li><li>Antony, E., Hingorani, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+ATP+binding+and+hydrolysis+activity+of+the+Thermus+aquaticus+MutS+dimer+is+key+to+modulation+of+its+interactions+with+mismatched+DNA.">Asymmetric ATP binding and hydrolysis activity of the Thermus aquaticus MutS dimer is key to modulation of its interactions with mismatched DNA.</a> Biochemistry. 43 (41), 13115-13128 (2004).</li><li>Gradia, S., Acharya, S., Fishel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+mismatch+recognition+complex+hMSH2-hMSH6+functions+as+a+novel+molecular+switch.">The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch.</a> Cell. 91 (7), 995-1005 (1997).</li><li>Mazur, D. J., Mendillo, M. L., Kolodner, R. D., D, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Msh6+ATPase+activity+by+mispaired+DNA+induces+a+Msh2(ATP)-Msh6(ATP)+state+capable+of+hydrolysis-independent+movement+along+DNA.">Inhibition of Msh6 ATPase activity by mispaired DNA induces a Msh2(ATP)-Msh6(ATP) state capable of hydrolysis-independent movement along DNA.</a> Mol Cell. 22 (1), 39-49 (2006).</li></ol>

一个DNA错配约束力这里描述的蛋白质的例子说明了电源的瞬态动力学方法研究生物分子机制的效用。停流单周转时间尺度上的测量提供了快速和具体MSH2，MSH6蛋白结合到一个不匹配的碱基对，并形成一个长寿命的蛋白质X DNA 复合物，在反应9确凿证据。此外，停流（淬流）ATP酶活性的分析提供了直接证据MSH2，MSH6开关约束力不匹配的 DNA 11,16后的ATP绑定构象。假设此开关信号DNA修复<su...

<table border="1"><tr><td> DNA的名称 </td><td> 序列 </td></tr><tr><td> 37摹</td><td> 5&#39; - ATT台泥TTC AGC AGA达摹电讯管理局局长文建会交咨会TGA TTC ACA的T -3“ </td></tr><tr><td> 37 T（2 - AP） </td><td> 5&#39; - ATG TGA ATC AGT ATG的多伦多T 鸭 T ATC TGC TGA AGG的AAA级T -3“ </td></tr><tr><td> 37 T </td><td> 5&#39; - ATG TGA ATC AGT ATG的多伦多T A T ATC TGC TGA AGG的AAA级T -3&#39; </td></tr></table>

application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein

瞬态动力学分析为了解生物大分子的工作是必不可少的，因为这种方法产生机理的信息，包括执政大分子功能的活性部位的浓度和内在的速率常数。例如，在酶的情况下，瞬时或稳定状态测量识别和描述反应途径中的个别事​​件，而稳态测量不仅产量整体的催化效率和特异性。蛋白质 - 蛋白质或蛋白质 - 配体相互作用的构象变化和速率限制，例如个别的事件经常发生在毫秒时间尺度，并可以直接停流和化学淬流方法进行测量。作为一个强大和方便的工具，从基板监测反应进程绑定到产品发布和催化营业额1,2的考虑，如荧光光信号，停止流。 在这里，我们报告的停流动力学的应用程序来探测MSH2，MSH6的作用机制，真核生物的DNA修复蛋白识别碱基错配和DNA和信号失配修复 （MMR）3-5插入/缺失循环。这样做，MSH2，MSH6提高三个数量级（错误的频率降低〜10 -6至10 -9基地）的DNA复制的准确性，从 ​​而有助于维护基因组的完整性。毫不奇怪，MSH2，MSH6功能有缺陷的人类与遗传性非息肉性结肠癌和其他零星的癌症 6-8 。为了了解这个关键的DNA代谢蛋白质的作用机制，我们正在调查MSH2，MSH6互动的动态与不匹配的DNA以及ATP酶的活性，燃料，在MMR其行动。 DNA结合迅速用含有2 - 氨基嘌呤（2 - AP）荧光基团相邻的G DNA混合MSH2，MSH6：T错配和监测在2 - 氨基嘌呤的荧光增加实时测量。 DNA解离测量混合预先形成MSH2，MSH6 G：T（2 - AP）的不匹配，与未标记的陷阱的DNA和复杂的监测荧光减少， 随着时间的推移9。测量荧光的变化7 - 二乙氨基-3 - （（（（2 maleimidyl）乙基）氨基）羰基）香豆素）标记的磷酸盐结合蛋白磷酸具有约束力（MDCC PBP）（预稳态ATP酶动力学PI）公布MSH2，MSH6 ATP水解9,10。 数据显示MSH2，MSH6快速绑定到一个G：T错配，形成一个长寿命的MSH2，MSH6摹：T复杂，在抑制ATP水解的蛋白质稳定在一个ATP绑定的形式转结果。反应动力学为这一假设提供了明确的支持，ATP绑定MSH2，MSH6信号约束力的双螺旋结构不匹配的碱基对的DNA修复。 F.诺亚比罗和杰翟本文同样。

Watch this Scientific Journal Video about Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein at JoVE.com

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

MSH2，MSH6负责启动DNA复制错误的修复。在这里，我们目前的走向理解的瞬态动力学方法，这个关键的蛋白质是如何工作的。该报告说明了停流实验测量耦合的DNA结合和ATP酶动力学的基本MSH2，MSH6在DNA修复中的作用机制。

停流的动力学方法的应用研究的一种DNA修复蛋白的行动机制

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

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Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

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Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

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Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

成像错配修复和DNA损伤的细胞反应枯草芽孢杆菌</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

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Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

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正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

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应用的<em在体外</em&gt;保护DNA检测，以可视化应力调解属性的DPS蛋白质

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

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Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

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Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...