Abstract：
Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5\' side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2\'-deoxyuridine > α-anomer of 2\'-deoxyadenosine >2\'-deoxyuridine > undamaged DNA. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme-substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes.
摘要：
来自大肠杆菌的嘌呤/无嘧啶（AP）内切核酸酶Nfo识别DNA中的AP位点，并催化AP位点5'侧的磷酸二酯键切割和一些受损或未受损的核苷酸。这里,通过脉冲电子-电子双共振(PELDOR，也称为DEER）光谱学和具有Frster共振能量转移检测DNA结合过程中DNA构象变化的预稳态动力学分析。模型DNA底物中靶核苷酸的核酸内切切割效率排名为（2R，3S)-2-(羟甲基)-3-羟基四氢呋喃[F位点]>5,6-二氢-2'-脱氧尿苷>2'-脱氧腺苷的α端基异构体>2'-脱氧尿苷>未受损DNA。在与Nfo相互作用期间DNA的实时构象变化表明，在初始酶-底物复合物的形成过程中，双链体末端之间的距离增加。在DEER测量中使用刚性接头自旋标记的DNA双链体表明，靶核苷酸本身的双螺旋弯曲和解绕是导致Nfo不加区别地识别靶核苷酸的关键因素之一。结果首次表明，来自不同结构家族的AP核酸内切酶利用了一种常见的损伤识别策略，它可以在全球范围内与其他酶在DNA中搜索特定位点的机制整合在一起。
