DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction.
DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction.
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人皮瓣核酸内切酶 1 催化反应所需的底物构象变化的 DNA 和蛋白质要求。
DOI：
10.1074/jbc.m115.698993
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发表时间：
2016-04-08
期刊：
影响因子：
--
通讯作者：
Grasby JA
中科院分区：
文献类型：
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作者：
Algasaier SI;Exell JC;Bennet IA;Thompson MJ;Gotham VJ;Shaw SJ;Craggs TD;Finger LD;Grasby JA
人瓣核酸内切酶-1（hFEN 1）催化复制和修复过程中DNA连接处产生的单链瓣的基本去除。hFEN 1生物学功能必须精确控制，因此，蛋白质依赖于蛋白质和底物构象变化的组合作为反应的先决条件。这些包括在双链体-双链体连接处的底物弯曲和未配对的反应双链体末端转移到活性位点。当存在时，5′-瓣被认为在螺旋帽下穿过，限制了对具有游离5′-末端的瓣的体内反应。在这里，我们监测的FRET和DNA解配对使用2-氨基嘌呤激子对CD的DNA弯曲，以确定这些基板的构象变化的DNA和蛋白质的要求。DNA与hFEN 1以弯曲构象的结合独立于5′-瓣调节发生，并且不需要活性位点金属离子或保守活性位点残基的存在。对于将底物转移到活性位点存在更严格的要求。将易断裂的磷酸二酯置于活性位点需要存在二价金属离子、游离的5′-瓣（如果存在）、反应双链体末端的沃森-克里克碱基对以及酶螺旋帽的完整二级结构。易分裂磷酸的最佳定位还需要活性位点保守残基Tyr 40、Asp 181和Arg 100以及反应性双链体5′-磷酸。这些研究表明了FEN 1反应机制，其中连接被结合并且5′-瓣被穿线（当存在时），并且最终底物被转移到活性位点金属上，从而引发裂解。
Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.