Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine.
Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine.
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DOI：
10.1021/bi001665g
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发表时间：
2001-01
期刊：
影响因子：
2.9
通讯作者：
E. Rachofsky;Eleanore Seibert;J. Stivers;R. Osman;J. Ross
中科院分区：
文献类型：
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作者：
E. Rachofsky;Eleanore Seibert;J. Stivers;R. Osman;J. Ross
脱碱基位点是DNA中高度致突变的损伤，其作为修饰碱基的切除修复的中间体而出现。这些位点由损伤特异性DNA糖基化酶的作用产生，并通过脱嘌呤/脱嘧啶核酸内切酶的特异性活性转化为下游中间体。已经在晶体结构中观察到这两个家族中的酶对碱基位点DNA施加变形，包括DNA扭结和碱基翻转。基于这些明显的蛋白质诱导的变形，我们提出，改变构象和动力学的脱碱基位点可能有助于这些修复酶的特异性。先前，对腺嘌呤类似物2-氨基嘌呤（2AP）的与脱碱基位点相对的稳态荧光的测量表明，二价阳离子的结合可以诱导构象变化，其增加2AP对溶质淬灭的可及性[Stivers，J.T.（1998）Nucleic Acids Res.26，3837-44]。我们已经进行了时间分辨荧光实验来表征参与这种构象变化的状态。这些研究的解释是基于最近开发的模型归因于静态和动态荧光猝灭的2AP在DNA中的芳香堆积和碰撞相互作用与相邻的碱基，分别（见前一篇论文在这个问题上）。时间分辨荧光的结果表明，二价阳离子结合转移两种构象之间的脱碱基位点的平衡：一个“封闭”的状态，其特征在于短的平均荧光寿命和复杂的衰减动力学，和一个“开放”的状态，其特征在于单指数衰减与寿命近似的游离核苷。由于掺入DNA的2AP的寿命和强度衰减动力学主要对与相邻碱基的碰撞相互作用敏感，因此在开放状态下不存在动态猝灭强烈表明荧光碱基在这种构象中是螺旋外的。与此解释相一致，时间分辨猝灭研究表明，开放状态是访问由碘化钾的溶质猝灭，但关闭状态不是。当荧光碱基侧翼为5 '-和3'-胸腺嘧啶时，在脱碱基位点中观察到比在5 '-和3'-腺嘌呤的情况下更大的静态淬灭，表明2AP与前一序列中的相邻碱基更多地堆叠。这些结果意味着脱碱基位点的构象以序列依赖的方式变化。无碱基位点被胸腺嘧啶取代的未受损序列不表现出开放状态，并且与其受损同源物相比具有不同水平的静态和动态淬灭。这些结构和动力学上的差异可能是修复酶对脱碱基位点的高特异性亲和力的重要决定因素。
Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.