Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459.
Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal 23955, Saudi Arabia.
J Biol Chem. 2018 Mar 30;293(13):4792-4804. doi: 10.1074/jbc.RA117.001137. Epub 2018 Feb 9.
Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion ( disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.
解旋酶能够催化复制、修复和重组过程中形成的单链 DNA 发夹结构的切割，因此对于基因组的加工和稳定性至关重要。最近报道的与 DNA 结合的人源解旋酶 1（hFEN1）的晶体结构，为 DNA 和 hFEN1 的构象变化如何促进反应机制提供了新的见解。例如，以前在非催化条件下使用 Ca 进行的 DNA 构象的生化研究表明，5'-发夹：模板连接处的碱基错配是反应的重要步骤，但新的结构数据表明并非如此。为了阐明 DNA 变化在动力学机制中的作用，我们在 Mg 存在的条件下测量了 hFEN1 催化反应中的一系列瞬态步骤，从底物结合到产物释放。我们发现，尽管 hFEN1 以快速、扩散限制的速率结合并弯曲 DNA，但随后在活性位点周围的 DNA 中发生较慢的 Mg 依赖性构象变化对于 5'-发夹的切割是必需的和限速的。这些变化总体上由 5'-发夹：模板连接处的 2-氨基嘌呤的荧光报告，表明局部 DNA 扭曲（结构中观察到的碱基堆积破坏）与将 5'-发夹的切割磷酸二酯键定位在 hFEN1 活性位点中相关，从而控制催化。在催化机制中具有不同作用的 hFEN1 残基，包括结合金属离子（天冬氨酸 34 和天冬氨酸 181）、引导 5'-发夹穿过活性位点和结合切割磷酸（赖氨酸 93 和精氨酸 100）以及堆积在切割磷酸的 5'端的碱基（酪氨酸 40），都有助于这些限速构象变化，确保 5'-发夹的有效和特异性切割。
