title: Fluorescence-based incision assay for human XPF-ERCC1 activity identifies important elements of DNA junction recognition.
abstract: The structure-specific endonuclease activity of the human XPF-ERCC1 complex is essential for a number of DNA processing mechanisms that help to maintain genomic integrity. XPF-ERCC1 cleaves DNA structures such as stem-loops, bubbles or flaps in one strand of a duplex where there is at least one downstream single strand. Here, we define the minimal substrate requirements for cleavage of stem-loop substrates allowing us to develop a real-time fluorescence-based assay to measure endonuclease activity. Using this assay, we show that changes in the sequence of the duplex upstream of the incision site results in up to 100-fold variation in cleavage rate of a stem-loop substrate by XPF-ERCC1. XPF-ERCC1 has a preference for cleaving the phosphodiester bond positioned on the 3'-side of a T or a U, which is flanked by an upstream T or U suggesting that a T/U pocket may exist within the catalytic domain. In addition to an endonuclease domain and tandem helix-hairpin-helix domains, XPF has a divergent and inactive DEAH helicase-like domain (HLD). We show that deletion of HLD eliminates endonuclease activity and demonstrate that purified recombinant XPF-HLD shows a preference for binding stem-loop structures over single strand or duplex alone, suggesting a role for the HLD in initial structure recognition. Together our data describe features of XPF-ERCC1 and an accepted model substrate that are important for recognition and efficient incision activity.
authors.affiliation: Structural Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3LY, UK.
authors.forename: Maureen
authors.initials: M
authors.lastname: Bowles
authors.forename: John
authors.initials: J
authors.lastname: Lally
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Fadden
authors.forename: Stephane
authors.initials: S
authors.lastname: Mouilleron
authors.forename: Timothy
authors.initials: T
authors.lastname: Hammonds
authors.forename: Neil Q
authors.initials: NQ
authors.lastname: McDonald
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D053837:DNA Cleavage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005470:Fluorometry / Q000379:methods; D006801:Humans; D007700:Kinetics; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D004268:DNA-Binding Proteins; C095888:xeroderma pigmentosum group F protein; D004247:DNA; C071447:ERCC1 protein, human; D004720:Endonucleases