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

Rachid Rezqui, Roxane Lestini, Joëlle Kühn, Xenia Fave, Lauren McLeod, Hannu Myllykallio, Antigoni Alexandrou, Cedric Bouzigues

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number0113493
JournalPLoS One
Volume9
Issue number11
DOIs
StatePublished - Nov 20 2014

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Flaps
single-stranded DNA
Endonucleases
Deoxyribonuclease I
image analysis
nucleases
Imaging techniques
kinetics
Molecules
Kinetics
DNA
Proliferating Cell Nuclear Antigen
Substrates
Pyrococcus abyssi
Extremities
Clamping devices
DNA Repair Enzymes
Pyrococcus
Substrate Specificity
Catalysis

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging. / Rezqui, Rachid; Lestini, Roxane; Kühn, Joëlle; Fave, Xenia; McLeod, Lauren; Myllykallio, Hannu; Alexandrou, Antigoni; Bouzigues, Cedric.

In: PLoS One, Vol. 9, No. 11, 0113493, 20.11.2014.

Research output: Contribution to journalArticle

Rezqui, Rachid ; Lestini, Roxane ; Kühn, Joëlle ; Fave, Xenia ; McLeod, Lauren ; Myllykallio, Hannu ; Alexandrou, Antigoni ; Bouzigues, Cedric. / Differential interaction kinetics of a bipolar structure-specific endonuclease with DNA flaps revealed by single-molecule imaging. In: PLoS One. 2014 ; Vol. 9, No. 11.
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