Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase
Kinetic mechanism of damage site recognition and uracil flipping by Escherichia coli uracil DNA glycosylase
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DOI：
10.1021/bi9818669
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发表时间：
1999-01-19
期刊：
影响因子：
2.9
通讯作者：
Watanabe, KA
中科院分区：
文献类型：
--
作者：
Stivers, JT;Pankiewicz, KW;Watanabe, KA
DNA修复酶尿嘧啶DNA糖基化酶（UDC）通过将尿嘧啶碱基从DNA螺旋上翻转，催化DNA中诱变前尿嘧啶残基的N-糖苷键水解裂解。碱基翻转的机制以及这一步骤在位点特异性DNA结合和酶催化中所起的作用在很大程度上是未知的。在不存在糖苷键裂解的情况下，使用含有位于荧光核苷酸报告基团2-氨基嘌呤（2-AP）附近的2'-氟-2'-脱氧尿苷（U-β，U-α）的2'-α和2'-β氟异构体的底物类似物，研究了UDG与DNA结合和尿嘧啶翻转的热力学和动力学。活性测量表明，含有U-β或U-α核苷酸的DNA是UDG的慢10（7）倍的底物（t（1/2）约为20 h），这允许在不存在糖苷键裂解的情况下测量DNA结合和碱基翻转。当UDG结合这些类似物，但不结合其他DNA分子时，观察到4 - 8倍的2-AP荧光增强，正如预期的那样，由于相邻尿嘧啶的酶促翻转导致2-AP碱基堆积减少。热力学测量表明，UDG与dsDNA形成弱非特异性复合物（K-D（ns）= 1.5 μ M），并与含U-β的dsDNA结合紧密25倍（K-D（app）约为50 nM）。因此，碱基翻转对结合自由能的贡献小于约2 kcal/mol，并且不是UDG> 10（6）倍催化特异性的主要成分。在25 ℃下的动力学研究表明，位点特异性结合通过两步机制发生。第一步（E + S ES）涉及UDG与DNA的扩散控制结合，形成弱的非特异性复合物（K-D约为1.5 - 3 μ M）。第二步（ES E'F）涉及导致可逆尿嘧啶翻转的快速步骤（k（max）约为1200 s（-1））。该步骤之后紧接着是通过色氨酸荧光猝灭监测的UDG的构象变化。结果提供了证据的酶辅助尿嘧啶翻转的机制，并排除了被动的机制，其中酶的陷阱中的游离底物的瞬时extrahelical基地。数据表明，DNA的双链体结构在碱基翻转步骤之前局部不稳定，从而促进尿嘧啶的挤出。因此，碱基翻转对DNA结合的自由能贡献很小，但通过诱导配合机制对特异性贡献很大。
The DNA repair enzyme uracil DNA glycosylase (UDC) catalyzes hydrolytic cleavage of the N-glycosidic bond of premutagenic uracil residues in DNA by flipping the uracil base from the DNA helix. The mechanism of base flipping and the role this step plays in site-specific DNA binding and catalysis by enzymes are largely unknown. The thermodynamics and kinetics of DNA binding and uracil flipping by UDG have been studied in the absence of glycosidic bond cleavage using substrate analogues containing the 2'-alpha and 2'-beta fluorine isomers of 2'-fluoro-2'-deoxyuridine (U-beta, U-alpha) positioned adjacent to a fluorescent nucleotide reporter group 2-aminopurine (2-AP). Activity measurements show that DNA containing a U-beta or U-alpha nucleotide is a 10(7)-fold slower substrate for UDG (t(1/2) approximate to 20 h), which allows measurements of DNA binding and base flipping in the absence of glycosidic bond cleavage. When UDG binds these analogues, but not other DNA molecules, a 4-8-fold 2-AP fluorescence enhancement is observed, as expected for a decrease in 2-AP base stacking resulting from enzymatic flipping of the adjacent uracil. Thermodynamic measurements show that UDG forms weak nonspecific complexes with dsDNA (K-D(ns) = 1.5 mu M) and binds similar to 25-fold more tightly to U-beta containing dsDNA (K-D(app) approximate to 50 nM). Thus, base flipping contributes less than similar to 2 kcal/mol to the free energy of binding and is not a major component of the > 10(6)-fold catalytic specificity of UDG. Kinetic studies at 25 degrees C show that site-specific binding occurs by a two-step mechanism. The first step (E + S ES) involves the diffusion-controlled binding of UDG to form a weak nonspecific complex with the DNA (K-D approximate to 1.5-3 mu M). The second step (ES E'F) involves a rapid step leading to reversible uracil flipping (k(max) approximate to 1200 s(-1)). This step is followed closely by a conformational change in UDG that was monitored by the quenching of tryptophan fluorescence. The results provide evidence for an enzyme-assisted mechanism for uracil flipping and exclude a passive mechanism in which the enzyme traps a transient extrahelical base in the free substrate. The data suggest that the duplex structure of the DNA is locally destabilized before the base-flipping step, thereby facilitating extrusion of the uracil. Thus, base flipping contributes little to the free energy of DNA binding but contributes greatly to specificity through an induced-fit mechanism.