Biro F Noah, Zhai Jie, Doucette Christopher W, Hingorani Manju M
Molecular Biology and Biochemistry Department, Wesleyan University.
J Vis Exp. 2010 Mar 31(37):1874. doi: 10.3791/1874.
Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic information including active site concentrations and intrinsic rate constants that govern macromolecular function. In case of enzymes, for example, transient or pre-steady state measurements identify and characterize individual events in the reaction pathway, whereas steady state measurements only yield overall catalytic efficiency and specificity. Individual events such as protein-protein or protein-ligand interactions and rate-limiting conformational changes often occur in the millisecond timescale, and can be measured directly by stopped-flow and chemical-quench flow methods. Given an optical signal such as fluorescence, stopped-flow serves as a powerful and accessible tool for monitoring reaction progress from substrate binding to product release and catalytic turnover(1,2). Here, we report application of stopped-flow kinetics to probe the mechanism of action of Msh2-Msh6, a eukaryotic DNA repair protein that recognizes base-pair mismatches and insertion/deletion loops in DNA and signals mismatch repair (MMR)(3-5). In doing so, Msh2-Msh6 increases the accuracy of DNA replication by three orders of magnitude (error frequency decreases from approximately 10(-6) to 10(-9) bases), and thus helps preserve genomic integrity. Not surprisingly, defective human Msh2-Msh6 function is associated with hereditary non-polyposis colon cancer and other sporadic cancers(6-8). In order to understand the mechanism of action of this critical DNA metabolic protein, we are probing the dynamics of Msh2-Msh6 interaction with mismatched DNA as well as the ATPase activity that fuels its actions in MMR. DNA binding is measured by rapidly mixing Msh2-Msh6 with DNA containing a 2-aminopurine (2-Ap) fluorophore adjacent to a G:T mismatch and monitoring the resulting increase in 2-aminopurine fluorescence in real time. DNA dissociation is measured by mixing pre-formed Msh2-Msh6 G:T(2-Ap) mismatch complex with unlabeled trap DNA and monitoring decrease in fluorescence over time(9). Pre-steady state ATPase kinetics are measured by the change in fluorescence of 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin)-labeled Phosphate Binding Protein (MDCC-PBP) on binding phosphate (Pi) released by Msh2-Msh6 following ATP hydrolysis(9,10). The data reveal rapid binding of Msh2-Msh6 to a G:T mismatch and formation of a long-lived Msh2-Msh6 G:T complex, which in turn results in suppression of ATP hydrolysis and stabilization of the protein in an ATP-bound form. The reaction kinetics provide clear support for the hypothesis that ATP-bound Msh2-Msh6 signals DNA repair on binding a mismatched base pair in the double helix. F
瞬态动力学分析对于理解生物大分子的作用机制不可或缺，因为这种方法能产生包括活性位点浓度和控制大分子功能的内在速率常数等机制信息。例如，对于酶而言，瞬态或稳态前测量可识别并表征反应途径中的各个事件，而稳态测量仅能得出整体催化效率和特异性。诸如蛋白质 - 蛋白质或蛋白质 - 配体相互作用以及限速构象变化等单个事件通常发生在毫秒时间尺度内，可通过停流和化学淬灭流方法直接测量。鉴于诸如荧光等光学信号，停流是监测从底物结合到产物释放以及催化周转的反应进程的强大且易用的工具(1,2)。在此，我们报告了应用停流动力学来探究Msh2 - Msh6的作用机制，Msh2 - Msh6是一种真核生物DNA修复蛋白，可识别DNA中的碱基对错配以及插入/缺失环并发出错配修复（MMR）信号(3 - 5)。通过这种方式，Msh2 - Msh6将DNA复制的准确性提高了三个数量级（错误频率从约10(-6)降至10(-9)碱基），从而有助于维持基因组完整性。毫不奇怪，人类Msh2 - Msh6功能缺陷与遗传性非息肉病性结肠癌和其他散发性癌症相关(6 - 8)。为了理解这种关键的DNA代谢蛋白的作用机制，我们正在探究Msh2 - Msh6与错配DNA相互作用的动力学以及为其在MMR中的作用提供能量的ATP酶活性。通过将Msh2 - Msh6与在G:T错配相邻处含有2 - 氨基嘌呤（2 - Ap）荧光团的DNA快速混合，并实时监测由此产生的2 - 氨基嘌呤荧光增加来测量DNA结合。通过将预先形成的Msh2 - Msh6 G:T(2 - Ap)错配复合物与未标记的捕获DNA混合，并监测荧光随时间的减少来测量DNA解离(9)。通过7 - 二乙氨基 - 3 - （（（（2 - 马来酰亚胺基）乙基）氨基）羰基）香豆素标记的磷酸结合蛋白（MDCC - PBP）在结合Msh2 - Msh6在ATP水解后释放的磷酸（Pi）时荧光的变化来测量稳态前ATP酶动力学(9,10)。数据显示Msh2 - Msh6与G:T错配快速结合并形成长寿命的Msh2 - Msh6 G:T复合物，这反过来又导致ATP水解受到抑制且蛋白质以ATP结合形式稳定。反应动力学为ATP结合的Msh2 - Msh6在结合双螺旋中的错配碱基对时发出DNA修复信号这一假设提供了明确支持。F
