Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase.
Uncoupling of nucleotide flipping and DNA bending by the t4 pyrimidine dimer DNA glycosylase.
复制标题
t4 嘧啶二聚体 DNA 糖基化酶解偶联核苷酸翻转和 DNA 弯曲。
DOI：
10.1021/bi060802s
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发表时间：
2006
期刊：
影响因子：
2.9
通讯作者：
Lloyd,RStephen
中科院分区：
文献类型：
--
作者：
Walker,RandallK;McCullough,AmandaK;Lloyd,RStephen
噬菌体T4嘧啶二聚体糖基化酶（T4-Pdg）是一种碱基切除修复蛋白，其在暴露于紫外光的结果而形成的环丁烷嘧啶二聚体处切割DNA。T4-Pdg与底物DNA的共聚合表明，胸腺嘧啶-胸腺嘧啶（TT）二聚体中与5 '-胸腺嘧啶相对的腺苷翻转成螺旋外构象，DNA骨架在酶-底物（ES）复合物中扭结60°。为了检查T4-Pdg反应机制中的预催化事件的动力学细节，设计研究以分别评估核苷酸翻转和DNA弯曲。荧光腺嘌呤碱基类似物，2-氨基嘌呤（2-AP），放置在一个脱碱基位点类似物，四氢呋喃，表现出2.8倍的发射强度增加时，翻转在ES复合物。使用用于核苷酸翻转的2-AP荧光信号，确定konandkoff预稳态动力学测量。使用位于双链体DNA中寡核苷酸5 '端的荧光供体-受体对，通过荧光共振能量转移评估DNA弯曲。供体荧光团的荧光强度在ES复合物中被淬灭15%，这是由于在弯曲构象中DNA的标记末端之间的能量转移效率增加。弯曲信号的动力学分析揭示了比核苷酸翻转的解离速率快2.5倍的解离速率。这些结果表明，在ES复合物的形成中，核苷酸翻转步骤可以与DNA的弯曲解偶联。
Bacteriophage T4 pyrimidine dimer glycosylase (T4-Pdg) is a base excision repair protein that incises DNA at cyclobutane pyrimidine dimers that are formed as a consequence of exposure to ultraviolet light. Cocrystallization of T4-Pdg with substrate DNA has shown that the adenosine opposite the 5‘-thymine of a thymine−thymine (TT) dimer is flipped into an extrahelical conformation and that the DNA backbone is kinked 60° in the enzyme−substrate (ES) complex. To examine the kinetic details of the precatalytic events in the T4-Pdg reaction mechanism, investigations were designed to separately assess nucleotide flipping and DNA bending. The fluorescent adenine base analogue, 2-aminopurine (2-AP), placed opposite an abasic site analogue, tetrahydrofuran, exhibited a 2.8-fold increase in emission intensity when flipped in the ES complex. Using the 2-AP fluorescence signal for nucleotide flipping,konandkoffpre-steady-state kinetic measurements were determined. DNA bending was assessed by fluorescence resonance energy transfer using fluorescent donor−acceptor pairs located at the 5‘-ends of oligonucleotides in duplex DNA. The fluorescence intensity of the donor fluorophore was quenched by 15% in the ES complex as a result of an increased efficiency of energy transfer between the labeled ends of the DNA in the bent conformation. Kinetic analyses of the bending signal revealed an off rate that was 2.5-fold faster than the off rate for nucleotide flipping. These results demonstrate that the nucleotide flipping step can be uncoupled from the bending of DNA in the formation of an ES complex.