Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
Cell Rep. 2013 Jun 27;3(6):1785-94. doi: 10.1016/j.celrep.2013.05.001. Epub 2013 Jun 6.
Human flap endonuclease 1 (FEN1), one of the structure-specific 5' nucleases, is integral in replication, repair, and recombination of cellular DNA. The 5' nucleases share significant unifying features yet cleave diverse substrates at similar positions relative to 5' end junctions. Using single-molecule Förster resonance energy transfer, we find a multistep mechanism that verifies all substrate features before inducing the intermediary-DNA bending step that is believed to unify 5' nuclease mechanisms. This is achieved by coordinating threading of the 5' flap of a nick junction into the conserved capped-helical gateway, overseeing the active site, and bending by binding at the base of the junction. We propose that this sequential and multistep substrate recognition process allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. Such mechanisms would also ensure the protection of DNA junctions from nonspecific bending and cleavage.
人类 flap endonuclease 1（FEN1）是结构特异性 5'核酸内切酶之一，它在细胞 DNA 的复制、修复和重组中不可或缺。这些 5'核酸内切酶具有显著的统一特征，但在相对于 5'端连接的相似位置切割不同的底物。使用单分子Förster 共振能量转移，我们发现了一种多步机制，该机制在诱导被认为统一 5'核酸内切酶机制的中介-DNA 弯曲步骤之前，验证了所有底物特征。这是通过协调将切口连接体的 5' flap 穿入保守的盖帽-螺旋门控来实现的，同时监管活性位点，并通过结合连接体的底部来弯曲。我们提出，这种顺序的和多步的底物识别过程允许不同的 5'核酸内切酶识别不同的底物，并将 DNA 弯曲的诱导限制在最后一个共同步骤。这种机制还将确保 DNA 连接体免受非特异性弯曲和切割的影响。
