Direct observation of DNA threading in flap endonuclease complexes.
Direct observation of DNA threading in flap endonuclease complexes.
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DOI：
10.1038/nsmb.3241
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发表时间：
2016-07
影响因子：
16.8
通讯作者：
Artymiuk PJ
中科院分区：
文献类型：
--
作者：
AlMalki FA;Flemming CS;Zhang J;Feng M;Sedelnikova SE;Ceska T;Rafferty JB;Sayers JR;Artymiuk PJ
维持基因组的完整性需要精确地加工分支核酸分子以产生双螺旋DNA。瓣状核酸内切酶是在复制过程中修剪由冈崎片段合成产生的这种分支分子的必需酶。在这里，我们报告的晶体结构的噬菌体T5瓣核酸内切酶与完整的DNA基板，和产品的复合物，在1.9-2.2毫米的分辨率。它们揭示了单链DNA穿过酶中的一个孔，该孔被一个横跨活性位点的倒V形螺旋拱包围。在DNA底物中，排列在孔中的残基诱导了一种不寻常的类β构象，并将易分裂的磷酸盐和必需的催化金属离子并列。一系列的复合物和生化分析显示了底物的单链分支如何接近、穿过并最终出现在酶的远端。我们的研究表明，基板识别涉及一个不寻常的“飞铸造，线程，弯曲和倒钩”的机制。
Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “fly-casting, thread, bend and barb” mechanism.
影响因子：
14.9
作者：
Bayliss CD;Sweetman WA;Moxon ER
通讯作者：
Moxon ER