Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates.
Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates.
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DOI：
10.1093/nar/gkt1116
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发表时间：
2014-02
影响因子：
14.9
通讯作者：
Penedo JC
中科院分区：
文献类型：
--
作者：
Craggs TD;Hutton RD;Brenlla A;White MF;Penedo JC
Flap内切核酸酶1（Fen 1）是一种高度保守的结构特异性核酸酶，能催化双链DNA底物上的5′端活瓣被切除。Fen 1在关键的细胞过程中起着至关重要的作用，如DNA复制和修复，而损害Fen 1表达水平或活性的突变对人类健康有严重的影响。Fen 1和其他FEN家族成员的核酸酶活性可以通过持续合成能力钳（proliferating cell nuclear antigen，PCNA）来刺激;然而，PCNA激活的确切机制目前尚不清楚。在这里，我们使用了合奏和单分子Förster共振能量转移与蛋白质诱导的荧光增强的组合来解偶联和研究Fen 1和Fen 1/PCNA复合物的底物识别和催化步骤。我们提出了一个模型，其中Fen 1结合后，一个高度动态的基板被弯曲和锁定成一个开放的皮瓣构象，可以建立特定的Fen 1/DNA相互作用。PCNA通过进一步促进5′ ssDNA瓣的易化穿线步骤中的开放瓣构象来增强Fen 1对DNA底物的识别。将我们的数据与现有的晶体学和分子动力学模拟相结合，我们提供了一个基于溶液的Fen 1/PCNA/DNA三元复合物模型。
Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.
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DOI：
10.1073/pnas.121075598
发表时间：
2001-07-17
影响因子：
11.1
作者：
Debrauwère, H;Loeillet, S;Nicolas, A
通讯作者：
Nicolas, A
影响因子：
--
作者：
Finger, L David;Atack, John M;Shen, Binghui
通讯作者：
Shen, Binghui
影响因子：
11.4
作者：
Beattie, Thomas R.;Bell, Stephen D.
通讯作者：
Bell, Stephen D.
影响因子：
13.8
作者：
Grasby, Jane A.;Finger, L. David;Tsutakawa, Susan E.;Atack, John M.;Tainer, John A.
通讯作者：
Tainer, John A.
影响因子：
4.8
作者：
Kao, KI;Henricksen, LA;Bambara, RA
通讯作者：
Bambara, RA