title: PCNA and XPF cooperate to distort DNA substrates.
abstract: XPF is a structure-specific endonuclease that preferentially cleaves 3' DNA flaps during a variety of repair processes. The crystal structure of a crenarchaeal XPF protein bound to a DNA duplex yielded insights into how XPF might recognise branched DNA structures, and recent kinetic data have demonstrated that the sliding clamp PCNA acts as an essential cofactor, possibly by allowing XPF to distort the DNA structure into a proper conformation for efficient cleavage to occur. Here, we investigate the solution structure of the 3'-flap substrate bound to XPF in the presence and absence of PCNA using intramolecular Förster resonance energy transfer (FRET). We demonstrate that recognition of the flap substrate by XPF involves major conformational changes of the DNA, including a 90 degrees kink of the DNA duplex and organization of the single-stranded flap. In the presence of PCNA, there is a further substantial reorganization of the flap substrate bound to XPF, providing a structural basis for the observation that PCNA has an essential catalytic role in this system. The wider implications of these observations for the plethora of PCNA-dependent enzymes are discussed.
authors.affiliation: Centre for Biomolecular Sciences and School of Physics and Astronomy, University of St Andrews, North Haugh, St Andrews, KY16 9SS, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hutton
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Craggs
authors.forename: Malcolm F
authors.initials: MF
authors.lastname: White
authors.forename: J Carlos
authors.initials: JC
authors.lastname: Penedo
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C095888:xeroderma pigmentosum group F protein; D004247:DNA; D004706:Endodeoxyribonucleases