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J. Biol. Chem., Vol. 278, Issue 40, 38803-38812, October 3, 2003

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dUTPase and Nucleocapsid Polypeptides of the Mason-Pfizer Monkey Virus Form a Fusion Protein in the Virion with Homotrimeric Organization and Low Catalytic Efficiency^*

Orsolya Barabás  , Michaela Rumlová ¶ ||, Anna Erdei **, Veronika Pongrácz , Iva Pichová ¶ and Beáta G. Vértessy  

From the Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary, the Department of Theoretical Chemistry, Eötvös Loránd University, POB 32, H-1518 Budapest, Hungary, the ^¶Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic, the ^**Department of Immunology, Eötvös Loránd University, Budapest, Hungary, and the ^||Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn²⁺ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)₄ to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.

Received for publication, June 30, 2003 , and in revised form, July 15, 2003.

^* This work was supported by the Hungarian National Research Foundation (Grants T034120, TS044730, and M27852 (to B. G. V.) and T034944), the Howard Hughes Medical Institutes Grant 55000342 (to B. G. V.), the Alexander von Humboldt Foundation, the Aventis/Institut de France Scientia Europeae Prize (to B. G. V.), by Grant IAA405304 from the Grant Agency of the Academy of Sciences of the Czech Republic and Research project Z4055905 (to I. P.), and by Grant 223300006 supported by Czech Ministry of Education (to M. R.). 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: Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, POB 7, H-1518, Budapest, Hungary. E-mail: vertessy{at}enzim.hu.

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