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.

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea

“…NucS was reported to be a bipolar nuclease acting on branched DNAs, including flapped and splayed DNAs ( 27 ). Furthermore, extensive structural analyses of this protein have been published ( 30 , 31 ). Therefore, we tested the branched DNAs, as well as the mismatched DNAs, as substrates for TkoEndoMS.…”

“…Proliferating cell nuclear antigen (PCNA) is a well-known clamp molecule that provides a scaffold for many proteins functioning in DNA replication and repair ( 35 , 36 ). Pyrococcus abyssi NucS reportedly interacts with its cognate PCNA ( 27 , 30 , 31 ). The putative PCNA-binding sequence in TkoEndoMS is QKTLF, at the C terminus (Supplementary Figure S1), and it seems to be a short version of the PIP ( P CNA I nteracting P rotein) box ( 37 ).…”

Archaeal DNA Repair Mechanisms

“…To identify potential MMR enzymes in species apparently lacking MutL/MutS, cosmid-expressed Pyrococcus furiosus genome regions were screened for the ability to cleave the DNA backbone at the site of mismatches and resulted in the isolation of NucS/EndoMS [78]. NucS (later aliased EndoMS) constitutes a novel family of archaeal and bacterial endonucleases originally identified when bioinformatically screening the genome of Pyrococcus abyssii for an amino acid motif known to bind the replication clamp PCNA [79]. Initially, NucS/EndoMS was investigated for activity upon branched structures which arise due to DNA damage or as a replication intermediate, but not for recognition of mismatched bases [80].…”

New Insights Into DNA Repair Revealed by NucS Endonucleases From Hyperthermophilic Archaea

“…The cleavage of flapped and splayed DNA substrates by Pab NucS suggests that this endonuclease is a potential archaeal NER endonuclease that can remove helix-distorting DNA damages ( Rouillon and White, 2011 ). Furthermore, Rezgui et al (2014) revealed that NucS endonuclease is a bipolar structure-specific endonuclease. In vivo , the engineered NucS-knockout strains of S. acidocaldarius are sensitive to DNA adducts, however, they did not show higher mutation rates ( Suzuki and Kurosawa, 2019 ).…”