title: Observation of unpaired substrate DNA in the flap endonuclease-1 active site.
abstract: The structure- and strand-specific phosphodiesterase flap endonuclease-1 (FEN1), the prototypical 5'-nuclease, catalyzes the essential removal of 5'-single-stranded flaps during replication and repair. FEN1 achieves this by selectively catalyzing hydrolysis one nucleotide into the duplex region of substrates, always targeting the 5'-strand. This specificity is proposed to arise by unpairing the 5'-end of duplex to permit the scissile phosphate diester to contact catalytic divalent metal ions. Providing the first direct evidence for this, we detected changes induced by human FEN1 (hFEN1) in the low-energy CD spectra and fluorescence lifetimes of 2-aminopurine in substrates and products that were indicative of unpairing. Divalent metal ions were essential for unpairing. However, although 5'-nuclease superfamily-conserved active-site residues K93 and R100 were required to produce unpaired product, they were not necessary to unpair substrates. Nevertheless, a unique arrangement of protein residues around the unpaired DNA was detected only with wild-type protein, suggesting a cooperative assembly of active-site residues that may be triggered by unpaired DNA. The general principles of FEN1 strand and reaction-site selection, which depend on the ability of juxtaposed divalent metal ions to unpair the end of duplex DNA, may also apply more widely to other structure- and strand-specific nucleases.
authors: Department of Chemistry, Centre for Chemical Biology, Krebs Institute, University of Sheffield, Sheffield, S3 7HF, UK and EaStCHEM School of Chemistry and Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre, The University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
authors: L David
authors: LD
authors: Finger
authors: Nikesh
authors: N
authors: Patel
authors: Amanda
authors: A
authors: Beddows
authors: Long
authors: L
authors: Ma
authors: Jack C
authors: JC
authors: Exell
authors: Emma
authors: E
authors: Jardine
authors: Anita C
authors: AC
authors: Jones
authors: Jane A
authors: JA
authors: Grasby
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation
chemical_list: D015075:2-Aminopurine; D004247:DNA; D045585:Flap Endonucleases; C468317:FEN1 protein, human
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