Differential Interaction Kinetics of a Bipolar Structure- Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps

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 59 and 39-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 59 or 39 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.
Type de document :
Article dans une revue
PLoS ONE, Public Library of Science, 2014, pp.0113493. 〈10.1371/journal.pone.0113493〉
Liste complète des métadonnées

Littérature citée [37 références]  Voir  Masquer  Télécharger

https://hal-polytechnique.archives-ouvertes.fr/hal-01102502
Contributeur : Denis Roura <>
Soumis le : lundi 12 janvier 2015 - 20:41:12
Dernière modification le : mercredi 25 avril 2018 - 10:45:03
Document(s) archivé(s) le : lundi 13 avril 2015 - 11:20:40

Fichier

journal.pone.0113493.pdf
Fichiers éditeurs autorisés sur une archive ouverte

Identifiants

Collections

Citation

Rachid Rezgui, Roxane Lestini, Joëlle Künh, Xenia Fave, Lauren Mcleod, et al.. Differential Interaction Kinetics of a Bipolar Structure- Specific Endonuclease with DNA Flaps Revealed by Single-Molecule Imaging Differential Interaction Kinetics of a Bipolar Structure-Specific Endonuclease with DNA Flaps. PLoS ONE, Public Library of Science, 2014, pp.0113493. 〈10.1371/journal.pone.0113493〉. 〈hal-01102502〉

Partager

Métriques

Consultations de la notice

345

Téléchargements de fichiers

177