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J. Biol. Chem., Vol. 280, Issue 33, 29444-29453, August 19, 2005

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The Redox State of the Baculovirus Single-stranded DNA-binding Protein LEF-3 Regulates Its DNA Binding, Unwinding, and Annealing Activities^*

Victor S. Mikhailov¶, Kazuhiro Okano, and George F. Rohrmann

From the Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804 and the N. K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808, Russia

The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg²⁺-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 Cys²¹⁴ 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.

Received for publication, March 24, 2005 , and in revised form, May 23, 2005.

^* This work was supported by National Institutes of Health Grant GM9982536 (to G. F. R.) and by the Russian Foundation for Basic Research Grant 03-04-49126 (to V. S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Microbiology, Oregon State University, Nash Hall 220, Corvallis, OR 97331-3804. Tel.: 541-737-1794; Fax: 541-737-0496; E-mail: Victor.Mikhailov{at}orst.edu.

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