NucS Activity Studies at the Single Molecule Level
type: Dataset
contact point: Organization
  fn: CCSD
keyword: physphysphys-bio-phphysics-physicsphysics-physicsbiological-physics-physicsbio-ph
keyword: tirf-microscopy
keyword: pyrococcus-abyssi
keyword: reparation-enzymes
keyword: onde-evanescente
keyword: molecule-unique
keyword: theses
keyword: dna-protein-interaction-single-molecule
keyword: infoeu-reposemanticsdoctoralthesis
keyword: interaction-adnproteine
keyword: enzyme-de-reparation
landing page: https://theses.hal.science/tel-00842766
description: As DNA reparation enzymes are essential to preserve genome integrity, understanding the regulation pathway dynamics is crucial. Pyrococcus Abyssi NucS is a recently discovered nuclease acting on branched DNA substrates with free ssDNA extremities, called ﬂaps. The biochemical characterization of NucS has shown a nanomolar aﬃnity for both 5' and 3' ﬂaps with the existence of two DNA binding sites (site I and II) as well as a bidirectional activity characterized by the ability to cleave both 5 'and 3 ' ﬂaps. The mechanisms that are responsible for this activity, particularly its association and dissociation modalities, however, remain poorly understood. In order to probe the dynamics of NucS interactions with its substrate, we set up a total internal reﬂection ﬂuorescence microscope that allows the detection and tracking of single interaction events between ﬂuorescently labeled NucS and DNA substrates tethered to a glass surface. Using Quantum dots, we furthermore developed a new colocalization technique based on multilateration that allows for drift correction and colocalization with an accuracy of 50 nm. In our experiments, we investigated the association and dissociation kinetics of the protein-DNA complex and studied the mechanism that regulates the activity of the NucS at diﬀerent conditions. First, we report a bidirectional and oriented binding on 3' and 5' ﬂaps and reveal that NucS/DNA dissociation follows a mechanism that is not dependent on NucS diﬀusion on ssDNA towards the junction but rather by the NucS/ssDNA energy landscape. We moreover demonstrated a central role of salt concentration in the modulation of NucS/DNA interactions. Second, we show that the dissociation mechanism follows single step process with one rate limiting dissociation constant and conclude that NucS can stochastically unbind its substrate in non cleavage conditions at 23 ◦ C. Thereafter, we studied the association and dissociation kinetics of the NucS-DNA complex at 45 ◦ Cin the pres…
issued: 2026-05-10T10:12:33.816196
language: fr
modified: 2026-05-10T10:12:33.816201

test_moissonnage_selune
type: Agent

NucS Activity Studies at the Single Molecule Level
type: Distribution
access URL: https://theses.hal.science/tel-00842766
format: HTML
issued: 2026-05-10T10:12:33.824047
modified: 2026-05-10T10:12:33.803631