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毫克金额11,12和类似的配体可以编写或购买。 <ol><li>我们过度表达S.在大肠杆菌中的酵母MSH2 - MSH6 大肠杆菌和纯化蛋白质离子交换色谱法（图1）11毫克量。 </li><li>可以通过化学方法合成2 - 氨基嘌呤的荧光基带或不带DNA试​​剂。我们购买了37个核苷酸长度的单链的DNA与2 - 氨基嘌呤修改和退火两种互补链准备双链DNA与2 - 氨基嘌呤毗邻的G：T错配（图2）。可以由制造商或在实验室纯化的DNA变性聚丙烯酰胺凝胶电泳;后者通常提供更高的产量。 </li><li> DNA结合动力学，准备单独的G：T（2 - AP）的DNA和DNA结合缓冲液中MSH2，MSH6蛋白样品（20毫米的Tris - HCl，pH值8.0，氯化镁2 5毫米，100毫米氯化钠）。 MSH2，MSH6和DNA浓度分别为0.8微米和0.12μM，这将产生0.4微米，0.06微米终浓度在一个1:1的混合比的混合实验。对于DNA解离动力学，准备一个样本含有0.8微米MSH2，MSH6和0.12微米G：T（2 - AP）的DNA，而另一个未标注摹8微米的样本：T DNA作为一个免费MSH2，MSH6的陷阱。准备和储存在冰样本。在这些反应中，蛋白质浓度MSH2，MSH6 - DNA的相互作用及DNA浓度远高于10 纳米 K ð足够强大的荧光信号（凭经验确定） 。 400个样品液的体积是足以获得约10（见表1）动能的痕迹。 </li><li>使用高品质的化学品，无荧光的蛋白质，DNA，和缓冲区准备杂质。过滤0.2微米的膜，以避免堵塞粒子停流的所有缓冲区。 </li><li>如果荧光基团吸收可见光，准备和实验，必须在低光照条件下进行。 </li></ol> 2。 MSH2，MSH6 DNA结合动力学的仪器准备一个停流的仪器是很简单的原则。迅速推入一个搅拌装置在驱动器注射器的两个解决方案，它使用驱动马达的混合溶液，然后流入数据收集（图3）观察细胞。我们使用的KinTek停流，这就需要样品量低，允许单一或连续搅拌反应物，各种光信号的检测，是非常容易使用。停流仪器以及其他几个厂家提供。 <ol><li>为了准备实验的停流仪器，确保计算机和控制器处于关闭状态。设置环境温度的循环水浴（25℃，C），打开冷却灯泡。点燃的灯。设置单色到所需的激发波长（315 nm）的2 - 氨基嘌呤。狭缝轮转动所需的狭缝宽度（经验平衡与低光漂白的荧光团激发高）。打开控制器和计算机。打开循环水浴保持在所需温度（30 ° C的情况下酿酒酵母 MSH2，MSH6）在实验过程中的反应物来填补水套。 </li><li>接下来，执行停流方案。打开选择的探测器，在这种情况下是干扰滤波器的光电倍增管（PMT）的荧光（350 nm的2 - 氨基嘌呤的情况下切断过滤器）的适当。施加电压的光电倍增管。暗电流测量，以纠正任何背景的电气噪声。 </li><li>停流驱动器的注射器和观察细胞装载样品前必须洗净。设置上样阀的负载位置。一个1ml注射器装满去离子水，过滤后的水，将它的装货港位于驱动器的注射器下方，并手动推动两者之间的注射器几次水。两次重复的过程中，为每个驱动器在实验中使用的注射器。下一步，填写驱动器与水的注射器冲洗观察细胞。样品装载阀切换到消防的位置。使用调整的注射器驱动器命令，该命令，控制，驱动电机，和较低的驱动板，通过观察细胞和进入退出行推水。小心不要推到驱动器注射器的柱塞。提高的驱动板。阀门开关的负载位置。手动用反应缓冲液的注射器，并保留为空。该仪器是目前投入使用。 </li></ol> 3。 MSH2，MSH6 DNA结合实验和数据分析<ol><li>每个样品管转移到一个新的1 ml注射器。每个样品注射器附加到装货港和解决方案的驱动器注射器推入。小心手动推动两者之间的注射器与间歇几次暂停的解决方案，以消除所有气泡。下驱动板，直到它接触的驱动器注射器的顶端。让反应物平衡的环境温度为几分钟。 </li><li>数据收集，打开设置时间/通道“窗口，选择数据收集渠道（PMT），分析数据模式（荧光），收集的痕迹（射击）。输入数据的收集，将收集的时间，在此期间，1000个数据点。一个未知的反应，应监测超过几秒钟，以便及时发现任何慢的阶段。凭经验估计达到平衡或稳定状态和设置数据采集时间≥6的一半生活所需的时间。在MSH2，MSH6的情况下的最佳时间框架是2秒为G：T错配结合动力学和60秒为G：T解离动力学。现在设置阀门，消防位置，然后单击“收集数据”按钮。该行动启动搅拌反应MSH2，MSH6和DNA，进入观察细胞，2 - 氨基嘌呤的荧光团激发和收集荧光信号随着时间的推移。收集至少5动能的痕迹，以获得高质量的数据，并保存该文件。 </li><li>当实验完成后，打开灯泡。如前所述，用清水洗净注射器和观察细胞。然后，关闭其他的设备，除用于循环水浴。水是一个额外的15分钟冷却灯泡散发。 </li><li>数学运算和数据拟合，可以进行与KinTek软件本身，以获得在实验过程中的反应动力学感。进一步分析还可以平均多动能的痕迹，保存和其他数据分析/制图软件出口的平均文件执行。 </li></ol> 4。 MSH2，MSH6 DNA结合动力学的代表成果与G MSH2，MSH6相互作用的动力学数据：T错配的DNA，可以适合一个单一的指数函数和快速的具有约束力的速率 常数 k的收益率接近3 × 10 71秒的M - 1（图4A）和缓慢解离常数 k 关 0.012的第二个-1（图4B），这表明，MSH2，MSH6绑定G：T错配的迅速形成一个非常稳定的复合物的半衰期接近60 秒 13 。 B.测量MSH2，MSH6 ATP酶动力学 1。 MSH2，MSH6 ATP酶动力学实验样品制备<ol><li>准备MSH2，MSH6和DNA试剂如前面所述，使用未标记的DNA。此外，净化大肠杆菌的磷酸盐结合蛋白（PBP ） MDCC荧光团（图5）14,15 大肠杆菌和标签。 MDCC - PBP结合自由磷酸盐迅速（ON等于1 × 8 M -1第二-1 K），并具有高亲和力（K = 100纳米），导致MDCC荧光（图6）在急剧增加。 MDCC - PBP因此，一个强大的记者稳态ATP水解和磷酸盐的释放动力学14,15。请注意，它是必不可少的，以尽量减少对这个实验的背景磷酸盐水平的，因此，使用的玻璃应严格避免在试剂准备的各个阶段。 </li><li>准备4微米MSH2，MSH6蛋白或无6微米摹样本：T DNA，从而产生2微米，3微米的最终浓度在一个1:1的混合比的混合实验。请注意，前稳态实验要求高酶浓度，因为感兴趣的信号从一个单一的营业额或前几催化失误。准备另一个样本含1毫米的新鲜溶解ATP和20微米的MDCC - PBP记者。新增0.10单位/ ml的嘌呤核苷磷酸化酶和200微米7 methylguanosine的样本，旨在扫荡任何污染磷酸。孵育至少20分钟（见表2）冰样本。 </li></ol> 2。 MSH2，MSH6 ATP酶动力学的仪器准备<ol><li>如前面所述，准备实验的停流仪器。此外，从驱动器注射器0.5单位/ ml嘌呤怒江拖把磷酸盐污染物cleoside磷酸和200微米7 methylguanosine 20分钟。激发波长425纳米和450 nm的截止滤光片使用MDCC PBP荧光检测的光电倍增管。 </li></ol> 3。 MSH2，MSH6 ATP酶活性实验和数据分析<ol><li>如前面所述的样品装入驱动器注射器。点击上收集数据，与ATP和MDCC - PBP和监控释磷混合MSH2，MSH6和加之随着时间的推移增加MDCC - PBP荧光MSH2，MSH6。收集获得一个高质量的数据集，并将该文件保存至少5动能痕迹。平均多动能痕迹和出口的数据文件进行分析。 </li><li>准备校准曲线，以确定之间的磷酸盐浓度和MDCC PBP荧光的线性关系。为了做到这一点，混合停流在相同的实验条件下不同的磷酸盐浓度，并测量最大MDCC PBP荧光每个磷酸盐浓度MDCC - PBP。 </li><li>剧情与每个磷酸盐浓度的校准曲线（图7）的最大MDCC PBP荧光和使用的斜坡，以获得在MSH2，MSH6反应磷酸盐浓度。剧情磷酸盐浓度随时间和数据拟合指数和线性函数。此功能描述一阵稳态ATP水解反应的线性稳态阶段释磷。 </li></ol> 4。 MSH2，MSH6 ATP酶动力学的代表成果动力学数据显示，MSH2，MSH6水解ATP并释放磷酸盐迅速在1.4第二催化第一成交量-1。这个阶段是在限制随后的失误，以7倍慢稳态0.2秒-1（图8A）k 猫的反应，其次是慢了一步。然而，当MSH2，MSH6势必不匹配的DNA，一阵水解ATP和磷酸释放被抑制，并MSH2，MSH6保持一个较长时间的ATP结合状态。 <img src="/files/ftp_upload/1874/1874fig1.jpg" alt="图1" /> 图1 S 的纯化。 从 E 酵母 MSH2，MSH6 大肠杆菌。MSH2和MSH6基因分别克隆到pET11a载体，并在大肠杆菌中表达大肠杆菌 BL21（DE3）细胞。 SP -琼脂糖凝胶，肝素，和Q -琼脂糖树脂柱层析纯化的蛋白复合物。 SDS - PAGE分析表明这里包含一个Q -琼脂糖柱的蛋白质组分。 <img src="/files/ftp_upload/1874/1874fig2.jpg" alt="图2" /> 图2的互补单链的DNA退火形成一个双面含G：T错配与相邻的腺苷（ATP酶实验）或2 -氨基嘌呤荧光基地模拟腺苷（DNA结合实验） 。 <img src="/files/ftp_upload/1874/1874fig3.jpg" alt="图3" /> 图3反应物流在KinTek停流在一个单一的混合实验。 <img src="/files/ftp_upload/1874/1874fig4a.jpg" alt="图4a" /> 图4A动力学MSH2，MSH6互动的G：T错配。双链DNA，包含一个G（0.12μM）混合：T相邻的2 -氨基嘌呤与MSH2，MSH6（0.8微米），导致荧光随着时间的推移增加，产量双分子速率常数k = 2.4 10 7 M -1小号-1的互动。 <img src="/files/ftp_upload/1874/1874fig4b.jpg" alt="图4b" /> 图4B动力学MSH2，MSH6互动的G：T错配。混合预先形成MSH2，MSH6 G：T（2 - AP）多余的未标记的摹复杂：T DNA（8微米），捕获任何自由MSH2，MSH6导致荧光随时间而减少，并产生一个缓慢的解离速率常数， k OFF = 0.012 s - 1时，表示与稳定，半衰期长的〜60秒复杂。终浓度：0.4μMMSH2，MSH6，0.06微米标记的DNA，4μm的未标记的摹：T DNA陷阱。 <img src="/files/ftp_upload/1874/1874fig5.jpg" alt="图5" /> 图5。MDCC PBP准备。 SDS - PAGE分析的大肠杆菌大肠杆菌磷酸结合蛋白（PBP），纯化的MDCC荧光基团标记。 <img src="/files/ftp_upload/1874/1874fig6.jpg" alt="图6" /> 图6。MDCC PBP响应磷酸盐。 MDCC - PBP滴定法磷酸（PI）增加MDCC荧光结果。 <img src="/files/ftp_upload/1874/1874fig7a.jpg" alt="图7a" /> 图7a。制备的磷酸（PI）的标准曲线。 MDCC - PBP（20μM）混合数额不等的PI（0 - 8微米）ð荧光测定随着时间的推移，直到达到平衡。终浓度：10μMMDCC的PBP，0 - 4微米丕。 <img src="/files/ftp_upload/1874/1874fig7b.jpg" alt="图7b" /> 图7b。磷酸盐的制备（PI）的标准曲线。最大MDCC PBP荧光绘制与 [PI]收益率的标准曲线。线的斜率（0.383μM-1在这种情况下）是用来转换成郫浓度MDCC - PBP荧光。 <img src="/files/ftp_upload/1874/1874fig8a.jpg" alt="图8a" /> 图8a。MSH2，MSH6 ATP酶的动力学。 MSH2，MSH6（4微米）的情况下摹，T的DNA，迅速与ATP（1毫米）和MDCC - PBP（20微米）混合展品一阵水解ATP和Pi释放。数据分析收益率（K = 1.4秒-1 水解 ）和振幅（2微米; MSH2，MSH6 1％的网站）突发阶段，这是一个线性的稳定状态阶段，在小号率0.4μM - 1（k CAT = 0.2秒-1）。 <img src="/files/ftp_upload/1874/1874fig8b.jpg" alt="图8b" /> 图8b。MSH2，MSH6 ATP酶动力学。 G：T DNA的反应（6微米）的增加，抑制ATP水解的破灭，稳定在ATP结合状态的复杂。终浓度：2微米，500微米，3微米的DNA，ATP的MSH2，MSH6 10μMMDCC - PBP。突发动力学符合下列公式：[皮] = 0 E - K 吨 + VT，其中[皮]是磷酸盐浓度，一个是一阵振幅0，K是观测到的突发速率常数和V是速度线性相位（k 猫 = V / [MSH2，MSH6]）。 <table border="1"><tbody><tr><td>样品</td><td style="text-align:center;" colspan="3">蛋白质</td><td style="text-align:center;" colspan="3">的DNA </td></tr><tr><td>试剂</td><td>股票</td><td>工作</td><td>卷，微升</td><td>股票</td><td>工作</td><td>卷，微升</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> MSH2，MSH6 </td><td> 5微米</td><td> 0.8微米</td><td> 64 </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 2ApG：T </td><td> - </td><td> - </td><td> - </td><td> 10μM </td><td> 0.12μM </td><td> 4.8 </td></tr><tr><td>缓冲区</td><td> 10X </td><td> 1X </td><td> 40 </td><td> 10X </td><td> 1X </td><td> 40 </td></tr><tr><td> DDH 2 Ø </td><td> - </td><td> - </td><td> 296 </td><td> - </td><td> - </td><td> 355 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 共有 </td><td></td><td></td><td> 400 </td><td></td><td></td><td> 400 </td></tr></tbody></table>表1 DNA结合反应<table border="1"><tbody><tr><td>样品</td><td colspan="3" style="text-align:center;">蛋白质</td><td colspan="3" style="text-align:center;">蛋白质- DNA </td><td colspan="3" style="text-align:center;">三磷酸腺苷</td></tr><tr><td>试剂</td><td>股票</td><td>工作</td><td>卷，微升</td><td>股票</td><td>工作</td><td>卷，微升</td><td>股票</td><td>工作</td><td>卷，微升</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> MSH2，MSH6 </td><td> 20μM </td><td> 4微米</td><td> 80 </td><td> 20μM </td><td> 4微米</td><td> 80 </td><td> - </td><td> - </td><td> - </td></tr><tr><td> G：T </td><td> - </td><td> - </td><td> - </td><td> 100 </td><td> 6 </td><td> 24 </td><td> - </td><td> - </td><td> - </td></tr><tr><td>三磷酸腺苷</td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 50毫米</td><td> 1毫米</td><td> 8 </td></tr><tr><td> 7 - MEG </td><td> 250X </td><td> 1X </td><td> 1.6 </td><td> 250X </td><td> 1X </td><td> 1.6 </td><td> 250X </td><td> 1X </td><td> 1.6 </td></tr><tr><td> PNPase </td><td> 100X </td><td> 1X </td><td> 4 </td><td> 100X </td><td> 1X </td><td> 4 </td><td> 100X </td><td> 1X </td><td> 4 </td></tr><tr><td> PBP - MDCC </td><td> 150微米</td><td> 20μM </td><td> 53.3 </td><td> 150微米</td><td> 20μM </td><td> 53.3 </td><td> - </td><td> - </td><td> - </td></tr><tr><td>缓冲区</td><td> 10X </td><td> 1X </td><td> 40 </td><td> 10X </td><td> 1X </td><td> 40 </td><td> 10X </td><td> 1X </td><td> 40 </td></tr><tr><td> DDH 2 Ø </td><td> - </td><td> - </td><td> 221 </td><td> - </td><td> - </td><td> 197 </td><td> - </td><td> - </td><td> 346 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 共有 </td><td></td><td></td><td> 400 </td><td></td><td></td><td> 400 </td><td></td><td></td><td> 400 </td></tr></tbody></table>表2 ATP酶反应

<ol>
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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. 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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。

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

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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18.0K 次观看.  Wesleyan University. 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。

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

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

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

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

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

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

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

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

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

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 ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

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), ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

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

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

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 ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂

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

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

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

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

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

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 ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

表面拴DNA大分子的可视化与荧光蛋白的DNA结合肽

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学

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