title: In eukaryotic flap endonuclease 1, the C terminus is essential for substrate binding.
abstract: Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease with important functions in DNA replication and DNA repair. It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro. The PCNA interaction site is located close to the C terminus of Fen1 and is flanked by a conserved basic region of 35-38 amino acids in eukaryotic species but not in archaea. We have constructed two deletion mutants of human Fen1 that lack either the PCNA interaction motif or a part of its adjacent C-terminal region and analyzed them in a variety of assays. Remarkably, deletion of the basic C-terminal region did not affect PCNA interaction but resulted in a protein with significantly reduced enzymatic activity. Electrophoretic mobility shift analysis revealed that this mutant displayed a severe defect in substrate binding. Our results suggest that the C terminus of eukaryotic Fen1 consists of two functionally distinct regions that together might form an important regulatory domain.
authors.affiliation: Institut für Veterinärbiochemie, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Stucki
authors.forename: Z O
authors.initials: ZO
authors.lastname: Jónsson
authors.forename: U
authors.initials: U
authors.lastname: Hübscher
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D045585:Flap Endonucleases; D006801:Humans; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human