Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, Zhejiang 310029, China.
Department of Cancer Genetics and Epigenetics, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA.
Nucleic Acids Res. 2018 Nov 30;46(21):11315-11325. doi: 10.1093/nar/gky911.
Human flap endonuclease 1 (hFEN1) is a structure-specific nuclease essential for DNA replication and repair processes. hFEN1 has 5' flap removal activity, as well as gap endonuclease activity that is critical for restarting stalled replication forks. Here, we report the crystal structures of wild-type and mutant hFEN1 proteins in complex with DNA substrates, followed by mutagenesis studies that provide mechanistic insight into the protein-protein interactions of hFEN1. We found that in an α-helix forming the helical gateway of hFEN1 recognizes the 5' flap prior to its threading into the active site for cleavage. We also found that the β-pin region is rigidified into a short helix in R192F hFEN1-DNA structures, suppressing its gap endonuclease activity and cycle-dependent kinase interactions. Our findings suggest that a single mutation at the primary methylation site can alter the function of hFEN1 and provide insight into the role of the β-pin region in hFEN1 protein interactions that are essential for DNA replication and repair.
人类核酸内切酶 1（hFEN1）是一种结构特异性核酸内切酶，对于 DNA 复制和修复过程至关重要。hFEN1 具有 5' 侧翼切除活性，以及缺口内切酶活性，这对于重新启动停滞的复制叉至关重要。在这里，我们报告了野生型和突变型 hFEN1 蛋白与 DNA 底物复合物的晶体结构，随后进行的突变研究提供了 hFEN1 蛋白-蛋白相互作用的机制见解。我们发现，在形成 hFEN1 螺旋门的α-螺旋中，在 5' 侧翼进入活性位点进行切割之前，它会先识别 5' 侧翼。我们还发现，在 R192F hFEN1-DNA 结构中，β-销钉区域被刚性化为短螺旋，抑制了其缺口内切酶活性和与周期依赖性激酶的相互作用。我们的研究结果表明，在主要甲基化位点的单个突变可以改变 hFEN1 的功能，并深入了解 β-销钉区域在 hFEN1 蛋白相互作用中的作用，这些相互作用对于 DNA 复制和修复至关重要。
