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J. Biol. Chem., Vol. 280, Issue 34, 30282-30290, August 26, 2005

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Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer^*

Arkadiusz Bibillo, Daniela Lener, Alok Tewari, and Stuart F. J. Le Grice¶

From the Reverse Transcriptase Biochemistry Section, Resistance Mechanisms Laboratory, HIV Drug Resistance Program, NCI, Frederick, National Institutes of Health, Frederick, Maryland 21702-1201

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix H of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln²⁹⁰, Phe²⁹², Gly²⁹⁴, Asn²⁹⁷, and Tyr²⁹⁸ (the counterparts of HIV-1 RT Gln²⁵⁸, Leu²⁶⁰, Gly²⁶², Asn²⁶⁵, and Trp²⁶⁶, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr²⁹⁸ with primer nucleotide -3, Gly²⁹⁴ with primer nucleotide -4, and Asn²⁹⁷ with template nucleotide -6. Substitution of Ala for Gln²⁹⁰ was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Received for publication, March 4, 2005 , and in revised form, May 10, 2005.

^* This work performed in the Reverse Transcriptase Biochemistry Section was supported by the HIV Drug Resistance Program, Center for Cancer Research, NCI, Frederick, National Institutes of Health. 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.

Present address: Inst. de Génétique Moléculaire de Montpellier, CNRS UMR5535, IFR122, 1919 Rte. de Mende, F-34293 Montpellier, Cedex 5, France.

Present address: Duke University, Health Policy Certificates Program, Durham, NC 27708.

^¶ To whom correspondence should be addressed: NCI, Frederick, National Institutes of Health, Bldg. 535, Frederick, MD 21702-1201. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice{at}ncifcrf.gov.

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