title: DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction.
abstract: Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5'-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5'-terminiin vivo Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5'-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5'-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr(40), Asp(181), and Arg(100)and a reacting duplex 5'-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5'-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage.
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Sana I
authors.initials: SI
authors.lastname: Algasaier
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Exell
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Bennet
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Thompson
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Victoria J B
authors.initials: VJ
authors.lastname: Gotham
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Shaw
authors.affiliation: the DNA:Protein Interactions Unit, School of Biochemistry, University of Bristol, Bristol BS8 1TD,United Kingdom.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Craggs
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and.
authors.forename: L David
authors.initials: LD
authors.lastname: Finger
authors.affiliation: From the Centre for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, United Kingdom and j.a.grasby@sheffield.ac.uk.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Grasby
mesh_terms: D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D045585:Flap Endonucleases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity
chemical_list: D004247:DNA; D045585:Flap Endonucleases; C468317:FEN1 protein, human
keywords: DNA endonuclease; DNA repair; DNA replication; DNA-protein interaction; circular dichroism (CD); fluorescence resonance energy transfer (FRET); nucleic acid enzymology