Human flap endonuclease-1:: Conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site
Human flap endonuclease-1:: Conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site
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DOI：
10.1021/bi002100n
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发表时间：
2001-03-13
期刊：
影响因子：
2.9
通讯作者：
Dyer, RB
中科院分区：
文献类型：
--
作者：
Kim, CY;Park, MS;Dyer, RB
人皮瓣核酸内切酶-1 (FEN-1) 是结构特异性核酸内切酶家族的成员，是 DNA 复制和修复的关键酶。 FEN-1 识别 5'-襟翼 DNA 结构并将其切割，这是一种特殊的核酸内切酶功能，对于 DNA 复制过程中冈崎片段的加工以及从带切口的双链 DNA 底物修复 5' 端单链尾部至关重要。镁是核酸酶活性所需的辅助因子。我们使用傅里叶变换红外 (FTIR) 光谱来更好地了解 Mg2+ 和瓣 DNA 如何与人类 FEN-1 相互作用。 FTIR 光谱为 FEN-1 与底物 DNA 和 Mg2+ 相互作用引起的结构变化提供了三个全新的见解。首先，酰胺I振动带(1600-1700 cm(-1))的FTIR差异光谱揭示了底物DNA结合引起的FEN-1二级结构的变化。 FTIR 光谱的定量分析表明，DNA 结合后螺旋度增加了 4%，或者约 14 个残基从无序构象转变为螺旋构象。观察到残基在没有 DNA 的情况下是无序的，这强烈暗示了柔性环区域。向螺旋的转变还表明了一种锁定结合 DNA 周围柔性环区域的机制。这是涉及蛋白质二级结构变化的结合机制的第一个直接实验证据。其次，与 DNA 结合相反，Mg2+ 与野生型或非裂解 D181A 突变体结合后，FEN-1 的二级结构没有观察到变化。第三，FTIR 结果提供了直接证据（通过 1535 cm(-1) 处的羧酸配体带），表明 D181 不仅是人酶中 Mg2+ 的配体，而且在缺乏该配体的 D181A 突变体中不会发生 Mg2+ 结合。
Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant, Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.