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

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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