| Abstract
| - Endonuclease V (endo V) recognizes and cleaves deoxyinosine in deaminated DNA. Theseenzymatic 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 techniqueof fluorescence resonance energy transfer (FRET), we determined the single-molecule kinetics of endo Vin a catalytic cycle using a substrate of deoxyinosine-containing single-stranded DNA (ssDNA). The ssDNAwas labeled with TAMRA, a fluorescence donor, while the endo V was labeled with Cy5, a fluorescenceacceptor. The time lapses of FRET, resulting from the sequential association, recognition, and dissociationof the deoxyinosine by the endo V, were determined at 5.9, 14.5, and 9.1 s, respectively, in the presenceof 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, ascompared to that of Mg2+ alone, support the hypothesis that endo V has two metal binding sites to regulateits enzymatic activities.
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