The redox state of the baculovirus single-stranded DNA-binding protein LEF-3 regulates its DNA binding, unwinding, and annealing activities |
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Authors: | Mikhailov Victor S Okano Kazuhiro Rohrmann George F |
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Affiliation: | Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804. |
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Abstract: | The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg(2+)-independent unwinding of DNA duplexes and annealing of complementary DNA strands. The unwinding and annealing activities of LEF-3 appeared to act in a competitive manner and were determined by the ratio of protein to DNA. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted unwinding at oversaturation of DNA substrates. The LEF-3 binding to ssDNA and unwinding activity were sensitive to redox agents and were inhibited by oxidation of thiol groups in LEF-3 with 1,1'-azobis(N,N-dimethylformamide) (diamide) or by modification with the thiol-conjugating agent N-ethylmaleimide. Both oxidation and alkylation increased the dissociation constant of the interaction with model oligonucleotides indicating a decrease in an intrinsic affinity of LEF-3 for ssDNA. These results proved that free thiol groups are essential both for LEF-3 interaction with ssDNA and for DNA unwinding. In contrast, oxidation or modification of thiol groups stimulated the annealing activity of LEF-3 partially due to suppression of its unwinding activity. Treatment of LEF-3 with the reducing agent dithiothreitol inhibited annealing, indicating association of this activity with the oxidized protein. Thus, the balance between annealing and unwinding activities of LEF-3 was determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. An LEF-3 mutant in which the conservative cysteine Cys(214) was replaced with serine showed both a decreased binding to DNA and a reduced unwinding activity, thus indicating that this residue might participate in the regulation of LEF-3 activities. |
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