Functional analysis of the hinge region close to the PCNA-binding region of human FEN1

皮瓣核酸内切酶1（FEN 1）和增殖细胞核抗原（PCNA）在DNA复制和修复中起着至关重要的作用。FEN 1是一种结构特异性核酸内切酶，其识别由具有5 '-未退火瓣（5'-flap DNA）的双链DNA组成的分支DNA结构。PCNA作为一种DNA钳蛋白，与5 ′-flap DNA上的FEN 1结合，然后刺激其核酸酶活性。通过对人FEN 1和PCNA之间的复合物的晶体结构的分析，揭示了人FEN 1-PCNA相互作用的结构基础。FEN 1在<333><336>C-末端尾部的核心结构域和PCNA相互作用蛋白（PIP）基序之间具有一个短的连接区，该连接区包含小残基（-^ QGST^ -），并且该连接区充当铰链，其可能在转换其核酸酶活性中起作用。为了通过测定PCNA刺激FEN核酸酶活性的活性来研究该铰链区在与PCNA结合的FEN 1核酸酶活性方面的重要性， 关于我们 我们制备了连接区具有较长柔性序列的人FEN 1突变体（-GSGS-（GS 2）、-GSGSGS-（GS 3）、-GSGSGSGS-（GS4）、-GSGSGSGS-（GS 5）），并进行了flap内切酶分析和FEN 1-PCNA结合分析。两个以上的接头残基（GS 3，GS4，GS 5）的延伸导致PCNA刺激的降低，而每个突变体显示与野生型无PCNA相同的核酸酶活性。这些结果清楚地表明铰链区的长度在PCNA刺激FEN 1核心结构域向DNA底物的方向上的重要性。此外，由于已知FEN 1活性受盐浓度的影响很大，因此使用PCNA突变体研究了核酸酶活性与NaCl浓度之间的关系。FEN 1核酸酶活性的动力学测量结果表明，PCNA/FEN 1复合物的形成与NaCl浓度之间存在显着的相关性。少

Flap endonuclease 1(FEN1) and proliferating cell nuclear antigen (PCNA) play a crucial role in DNA replication and repair. FEN1 is a structure-specific endonuclease that recognizes a branched DNA structure consisting of a double-stranded DNA with 5'-unannealed flap(5'-flap DNA). PCNA acts as a DNA clamp protein and binds to FEN1 on 5'-flap DNA, then stimulates its nuclease activity. The structural basis of the human FEN1-PCNA interaction was revealed by analysis of the crystal structure of the complex between human FEN1 and PCNA. FEN1 possesses a short linker region containing small residues (-^<333>QGST^<336>-) between the core domain and the PCNA-interacting protein(PIP) motif in the C-terminal tail, and this linker region acts as a hinge, which may play a role in switching its nuclease activity. In order to investigate the importance of this hinge region in terms of the nuclease activity of FEN1 bound to PCNA by assaying the activity of PCNA to stimulate the nuclease activity of FEN … More 1, we performed mutation experiments against this hinge region of FEN1.The human FEN1 mutants with longer and flexible sequences in the linker region (-GSGS-(GS2), -GSGSGS-(GS3), -GSGSGSGS-(GS4), -GSGSGSGSGS-(GS5))were prepared, and flap endonuclease assays and FEN1-PCNA binding assays were performed. Extension of more than two linker residues (GS3, GS4, GS5)resulted in decreased stimulation by PCNA, while every mutant showed the same nuclease activity as wild type without PCNA. These results clearly indicate the importance of the length of hinge region in terms of stimulation by PCNA to direct the FEN1 core domain toward the DNA substrate. Furthermore, the relationship between the nuclease activity and NaCl concentration was investigated using PCNA mutants, since it was known that FEN1 activity was highly influenced by salt concentration. Results of kinetic measurements of nuclease activity of FEN1 suggested that there were significant correlations between the PCNA/FEN1 complex formation and the NaCl concentration. Less