Lesion Recognition and Cleavage by Endonuclease V:  A Single-Molecule Study

Abstract

Endonuclease V (endo V) recognizes and cleaves deoxyinosine in deaminated DNA. These enzymatic activities are precursors of DNA repair and are fueled by metal ions such as Ca2+ and Mg2+, with the former being associated with protein binding and the latter with DNA cleavage. Using the technique of fluorescence resonance energy transfer (FRET), we determined the single-molecule kinetics of endo V in a catalytic cycle using a substrate of deoxyinosine-containing single-stranded DNA (ssDNA). The ssDNA was labeled with TAMRA, a fluorescence donor, while the endo V was labeled with Cy5, a fluorescence acceptor. The time lapses of FRET, resulting from the sequential association, recognition, and dissociation of the deoxyinosine by the endo V, were determined at 5.9, 14.5, and 9.1 s, respectively, in the presence of Mg2+. In contrast, the process of deoxyinosine recognition appeared little affected by the metal type. The prolonged association and dissociation events in the presence of the Ca2+-Mg2+ combination, as compared to that of Mg2+ alone, support the hypothesis that endo V has two metal binding sites to regulate its enzymatic activities.

Crystal structure of E. coli endonuclease V, an essential enzyme for deamination repair

“…EcEndoV starts to form a shift with ssDNA-I or ssRNA-I at a molar ratio of 1:1 in the presence of 5 mM Ca 2+ , which presumably is the complex of EcEndoV bound with one molecule of ssDNA-I/ssRNA-I (complex I). The purpose of Ca 2+ usage is to inhibit the cleavage activities of EcEndoV 21 22 . With increasing amounts of the protein, a supershift (complex II) is also visible.…”

“…In addition, EcEndoV also cleaves the U, O, X, apurinic/apyrimidinic (AP) site, insertion/deletion mismatches (IDM), flaps, loops and pseudo-Y DNA structures, when Mn 2+ is supplied as a metal cofactor. Studies revealed that T. Maritima EndoV (TmEndoV) works via a catalytic and regulatory two-metal mechanism, where Mg 2+ or Mn 2+ is responsible for the cleavage while Ca 2+ stimulates substrate binding 21 22 . When only Ca 2+ is present, EcEndoV forms stable complexes with deoxyinosine-containing DNA, mismatches-containing DNA, flaps, pseudo-Y, fork, 3-way- and holiday junction DNA 23 .…”

Structure of human endonuclease V as an inosine-specific ribonuclease

“…Additionally, E. coli EndoV (EcEndoV) also recognizes apurinic/apyrimidinic (AP) sites, mismatches, insertions/deletions, flaps, pseudo-Y structures, loops and other deaminated DNA bases in vitro , 1996Gates & Linn, 1977;He et al, 2000;Rosnes et al, 2013). In bacteria, the incision of dI by EndoV is a twometal-dependent reaction and produces a stable intermediate complex consisting of EndoV and the nicked DNA Dalhus et al, 2009;Mi et al, 2011;Feng et al, 2006;Lin et al, 2007). A model for the cleavage has been proposed in which Ca 2+ induces the recognition of dI and Mg 2+ is required for DNA cleavage (Lin et al, 2007;Feng et al, 2006).…”

“…In bacteria, the incision of dI by EndoV is a twometal-dependent reaction and produces a stable intermediate complex consisting of EndoV and the nicked DNA Dalhus et al, 2009;Mi et al, 2011;Feng et al, 2006;Lin et al, 2007). A model for the cleavage has been proposed in which Ca 2+ induces the recognition of dI and Mg 2+ is required for DNA cleavage (Lin et al, 2007;Feng et al, 2006).…”

Functional Studies of DNA-Protein Interactions Using FRET Techniques

“…The main advantage of this fluorimetric assay when compared to more conventional biochemical techniques such as radiolabelingbased electrophoresis or ELISA-based techniques relies on its continuous character, so that the cleavage reaction can be monitored from the initial steps in real-time with no need for extensive sample handling. Following this approach, the kinetics of restriction endonucleases such as PaeR7 [32], EcoRV [33], S1 nuclease [34], and Endonuclease V [35] have all been quantified by FRET techniques.…”