title: Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.
abstract: As DNA repair enzymes are essential for preserving genome integrity, understanding their substrate interaction dynamics and the regulation of their catalytic mechanisms is crucial. Using single-molecule imaging, we investigated the association and dissociation kinetics of the bipolar endonuclease NucS from Pyrococcus abyssi (Pab) on 5' and 3'-flap structures under various experimental conditions. We show that association of the PabNucS with ssDNA flaps is largely controlled by diffusion in the NucS-DNA energy landscape and does not require a free 5' or 3' extremity. On the other hand, NucS dissociation is independent of the flap length and thus independent of sliding on the single-stranded portion of the flapped DNA substrates. Our kinetic measurements have revealed previously unnoticed asymmetry in dissociation kinetics from these substrates that is markedly modulated by the replication clamp PCNA. We propose that the replication clamp PCNA enhances the cleavage specificity of NucS proteins by accelerating NucS loading at the ssDNA/dsDNA junctions and by minimizing the nuclease interaction time with its DNA substrate. Our data are also consistent with marked reorganization of ssDNA and nuclease domains occurring during NucS catalysis, and indicate that NucS binds its substrate directly at the ssDNA-dsDNA junction and then threads the ssDNA extremity into the catalytic site. The powerful techniques used here for probing the dynamics of DNA-enzyme binding at the single-molecule have provided new insight regarding substrate specificity of NucS nucleases.
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Rachid
authors.initials: R
authors.lastname: Rezgui
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Roxane
authors.initials: R
authors.lastname: Lestini
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Joëlle
authors.initials: J
authors.lastname: Kühn
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Xenia
authors.initials: X
authors.lastname: Fave
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Lauren
authors.initials: L
authors.lastname: McLeod
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Hannu
authors.initials: H
authors.lastname: Myllykallio
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Antigoni
authors.initials: A
authors.lastname: Alexandrou
authors.affiliation: Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS (Centre National pour la Recherche Scientifique) UMR (Unité Mixte de Recherche) 7645, Inserm (Institut national de la santé et de la recherche médicale) U696, Palaiseau, France.
authors.forename: Cedric
authors.initials: C
authors.lastname: Bouzigues
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D045585:Flap Endonucleases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D038761:Photobleaching; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D043903:Pyrococcus abyssi / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014783:Viscosity
chemical_list: D019843:Archaeal Proteins; D004277:DNA, Single-Stranded; D018809:Proliferating Cell Nuclear Antigen; D008345:Manganese; D004247:DNA; D045585:Flap Endonucleases