Lesion recognition and cleavage by endonuclease V: a single-molecule study.
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.
Lin
Jun
J
Department of Physics and Astronomy, South Carolina Experiment Station, Room 219 Biosystems Research Complex, 51 New Cherry Street, Clemson University, Clemson, South Carolina 29634, USA.
Gao
Honghai
H
Schallhorn
Kathryn A
KA
Harris
Rebecca M
RM
Cao
Weiguo
W
Ke
Pu Chun
PC
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D007288:Inosine / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008954:Models, Biological; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D007288:Inosine; D043224:Deoxyribonuclease (Pyrimidine Dimer); C012271:deoxyinosine; D008274:Magnesium; D002118:Calcium