Base-flipping mutations of uracil DNA glycosylase: substrate rescue using a pyrene nucleotide wedge
Base-flipping mutations of uracil DNA glycosylase: substrate rescue using a pyrene nucleotide wedge
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DOI：
10.1021/bi026227j
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发表时间：
2002-09-17
期刊：
影响因子：
2.9
通讯作者：
Song, FH
中科院分区：
文献类型：
--
作者：
Jiang, YL;Stivers, JT;Song, FH
我们最近推出了一种新的底物拯救工具，用于研究尿嘧啶 DNA 糖基化酶 (UDG) 的酶促碱基翻转，其中将大体积的芘核苷酸楔形 (Y) 放置在双链 DNA 中的尿嘧啶对面（即 U/Y 对），从而将目标碱基预组织为螺旋外构象 [Jiang, Y. L., et al. 2017]。 (2001) J.Biol。化学。 276, 42347-54]。芘楔完全挽救了因去除天然 Leu191 楔而产生的大催化缺陷，大概模仿了该基团的推动和堵塞功能。在这里，我们采用芘拯救方法结合瞬态动力学方法来评估 UDG 的六个保守酶基团的功能作用，这些酶基团与“捏、推、插、拉”碱基翻转机制有关（参见本期的前一篇论文）。我们发现，与 U/A 对相比，U/Y 碱基对将 L191G 推动突变的受损位点识别的表观二阶速率常数提高了 45 倍，从而完全挽救了突变的动力学效应。值得注意的是，U/Y 对还允许 L191G 继续完成与正常 U/A 底物严格组成的构象对接步骤，并允许 UDG 的活性位点夹在螺旋外基底周围。因此，芘还起到Leu191侧链的堵塞作用。通过芘将尿嘧啶预组织成螺旋外构象，允许所有这些碱基翻转突变体进行扩散控制的损伤识别，并允许 UDG 构象变化与天然 U/A 碱基对的尿嘧啶翻转速率一样快。因此，芘楔形底物允许 UDG 通过锁钥匙机制而不是自然诱导配合机制来识别尿嘧啶。非天然芘碱基对可能提供一种通用策略来促进识别螺旋外碱基的其他酶的位点特异性靶向。
We recently introduced a new substrate rescue tool for investigating enzymatic base flipping by uracil DNA glycosylase (UDG) in which a bulky pyrene nucleotide wedge (Y) was placed opposite a uracil in duplex DNA (i.e., a U/Y pair), thereby preorganizing the target base in an extrahelical conformation [Jiang, Y. L., et al. (2001) J. Biol. Chem. 276, 42347-54]. The pyrene wedge completely rescued the large catalytic defects resulting from removal of the natural Leu191 wedge, presumably mimicking the pushing and plugging function of this group. Here we employ the pyrene rescue method in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the "pinch, push, plug, and pull" base-flipping mechanism (see the preceding paper in this issue). We find that a U/Y base pair increases the apparent second-order rate constant for damaged site recognition by L191G pushing mutation by 45-fold as compared to a U/A pair, thereby fully rescuing the kinetic effects of the mutation. Remarkably, the U/Y pair also allows L191G to proceed through the conformational docking step that is severely comprised with the normal U/A substrate, and allows the active site of UDG to clamp around the extrahelical base. Thus, pyrene also fulfills the plugging role of the Leu191 side chain. Preorganization of uracil in an extrahelical conformation by pyrene allows diffusion-controlled damage recognition by all of these base-flipping mutants, and allows the UDG conformational change to proceed as rapidly as the rate of uracil flipping with the natural U/A base pair. Thus, the pyrene wedge substrate allows UDG to recognize uracil by a lock-and-key mechanism, rather than the natural induced-fit mechanism. Unnatural pyrene base pairs may provide a general strategy to promote site-specific targeting of other enzymes that recognize extrahelical bases.