Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by human alkyladenine DNA glycosylase.
Kinetic mechanism for the flipping and excision of 1,N(6)-ethenoadenine by human alkyladenine DNA glycosylase.
复制标题
人烷基腺嘌呤 DNA 糖基化酶翻转和切除 1,N(6)-乙烯腺嘌呤的动力学机制。
DOI：
10.1021/bi9015082
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
O'Brien,PatrickJ
中科院分区：
文献类型：
--
作者：
Wolfe,AbigailE;O'Brien,PatrickJ
人烷基腺嘌呤DNA糖基化酶启动DNA中各种各样的烷基化和脱氨基嘌呤损伤的修复。在这项研究中，我们利用1，N6-乙烯腺苷（εA）病变的自然荧光，并报告结合，核苷酸翻转，碱基切除和产物释放的动力学分析。εA荧光的瞬时变化揭示了在水解步骤之前形成的两种不同复合物的存在。一个初始的识别复合物的形式迅速，其特征是部分破坏的病变基地的堆叠相互作用。随后，形成非常稳定的螺旋外复合物，其中εA损伤被AAG活性位点口袋中的相互作用强烈淬灭。我们的研究结果表明，DNA结合和碱基翻转发生在毫秒到秒的时间尺度上。N-糖苷键裂解要慢得多，发生在分钟的时间尺度上。脉冲追踪实验被用来证明，即使是紧密结合的εA底物，螺旋外复合物也不能完全被切除。然而，εA的翻转是非常有利的，我们计算出翻转的平衡常数是1300。这种动力学机制具有重要的生物学意义。首先，两步结合提供了区分受损和未受损核苷酸的多种机会。第二，快速平衡翻转机制最大化受损碱基对未受损碱基的特异性，因为未受损碱基通常形成比受损碱基更强的碱基对。最后，对于εA翻转的高度有利的平衡确保了εA去除独立于序列背景并且高效，尽管N-糖苷键水解的速率相对较慢。
Human alkyladenine DNA glycosylase initiates the repair of a wide variety of alkylated and deaminated purine lesions in DNA. In this study, we take advantage of the natural fluorescence of the 1,N6-ethenoadenosine (εA) lesion and report a kinetic analysis of binding, nucleotide flipping, base excision, and product release. The transient changes in the fluorescence of εA revealed the existence of two distinct complexes that are formed prior to the hydrolysis step. An initial recognition complex forms rapidly and is characterized by partial disruption of the stacking interactions of the lesioned base. Subsequently, a very stable extrahelical complex is formed in which the εA lesion is strongly quenched by interactions in the AAG active site pocket. Our results indicate that DNA binding and base flipping take place on the millisecond to second time scale. N-Glycosidic bond cleavage is much slower, taking place on the minute time scale. A pulse−chase experiment was used to demonstrate that even for the tightly bound εA substrate, the extrahelical complex is not fully committed to excision. Nevertheless, flipping of εA is highly favorable, and we calculate that the equilibrium constant for flipping is ∼1300. This kinetic mechanism has important biological implications. First, two-step binding provides multiple opportunities to discriminate between damaged and undamaged nucleotides. Second, a rapid equilibrium flipping mechanism maximizes specificity for damaged versus undamaged bases, since undamaged bases generally form stronger base pairs than damaged bases. Finally, the highly favorable equilibrium for flipping of εA ensures that εA removal is independent of sequence context and highly efficient despite the relatively slow rate of N-glycosidic bond hydrolysis.