Dataset Information

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.

SUBMITTER: Rezgui R

PROVIDER: S-EPMC4239081 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

Rezgui Rachid R Lestini Roxane R Kühn Joëlle J Fave Xenia X McLeod Lauren L Myllykallio Hannu H Alexandrou Antigoni A Bouzigues Cedric C

PloS one 20141120 11

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 N ...[more]

PMID: 25412080

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Project description:Recent experimental and computational developments have been pushing the limits of live-cell single-molecule imaging, enabling the monitoring of intermolecular interactions in their native environment with high spatiotemporal resolution. However, interactions are captured only for the labeled subset of molecules, which tends to be a small fraction. As a result, it has remained a challenge to calculate molecular interaction kinetics, in particular association rates, from live-cell single-molecule tracking data. To overcome this challenge, we developed a mathematical modeling-based Framework for the Inference of in Situ Interaction Kinetics (FISIK) from single-molecule imaging data with substoichiometric labeling. FISIK consists of (I) devising a mathematical model of molecular movement and interactions, mimicking the biological system and data-acquisition setup, and (II) estimating the unknown model parameters, including molecular association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of the model and data, we adapted the method of indirect inference for model calibration. We validated FISIK using a series of tests in which we simulated trajectories of diffusing molecules that interact with each other, considering a wide range of model parameters, and including resolution limitations, tracking errors, and mismatches between the model and the biological system it mimics. We found that FISIK has the sensitivity to determine association and dissociation rates, with accuracy and precision depending on the labeled fraction of molecules and the extent of molecule tracking errors. For cases where the labeled fraction is too low (e.g., to afford accurate tracking), combining dynamic but sparse single-molecule imaging data with almost-whole population oligomer distribution data improves FISIK's performance. All in all, FISIK is a promising approach for the derivation of molecular interaction kinetics in their native environment from single-molecule imaging data with substoichiometric labeling.

Dataset Information

Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

Publications

Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging.

Similar Datasets

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