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Originally published In Press as doi:10.1074/jbc.M910361199 on March 23, 2000

J. Biol. Chem., Vol. 275, Issue 26, 19759-19767, June 30, 2000
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Coupling Ribose Selection to Fidelity of DNA Synthesis
THE ROLE OF Tyr-115 OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REVERSE TRANSCRIPTASE*

Clara E. Cases-González, Mónica Gutiérrez-RivasDagger , and Luis Menéndez-Arias§

From the Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

The catalytic efficiency of incorporation of deoxyribonucleotides by wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was around 100-fold higher than for dideoxyribonucleotides, in Mg2+-catalyzed reactions, and more than 10,000-fold higher than for nucleotides having a 2'-hydroxyl group in Mg2+- and Mn2+-catalyzed reactions. Mutant RTs with nonconservative substitutions affecting Tyr-115 (Y115V, Y115A, and Y115G) showed a dramatic reduction in their ability to discriminate against ribonucleotides in the presence of both cations. However, selectivity of deoxyribonucleotides versus ribonucleotides was not affected in mutants Y115W and F160W. The substitution of Tyr-115 with Val or Gly had no effect on discrimination against dideoxyribonucleotides, but these mutants were less efficient than the wild-type RT in discriminating against cordycepin 5'-triphosphate. We also show that Tyr-115 is involved in fidelity of DNA synthesis, but substitutions at this position have different effects depending on the metal cofactor used in the assays. In Mg2+-catalyzed reactions, removal of the side chain of Tyr-115 reduced misinsertion and mispair extension fidelity, while opposite effects were observed in Mn2+-catalyzed reactions. Our results indicate that the aromatic side chain of Tyr-115 plays a role as a "steric gate" preventing the incorporation of nucleotides with a 2'-hydroxyl group in a cation-independent manner, while its influence on fidelity could be modulated by Mg2+ or Mn2+.

* This work was supported in part by Fondo de Investigación Sanitaria Grant 98/0054-01 (to L. M.-A.) and by an institutional grant of Fundación Ramón Areces to Centro de Biología Molecular "Severo Ochoa."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a predoctoral fellowship of Instituto de Salud Carlos III.

§ To whom correspondence should be addressed. Tel.: 34-913978477; Fax: 34-913974799; E-mail: lmenendez@cbm.uam.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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