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J Biol Chem, Vol. 275, Issue 18, 13879-13887, May 5, 2000

Interaction of p55 Reverse Transcriptase from the Saccharomyces cerevisiae Retrotransposon Ty3 with Conformationally Distinct Nucleic Acid Duplexes*

Jason W. RauschDagger §, Marion K. Bona-Le Grice§, M. Henrietta, Nymark-McMahon||, Jennifer T. MillerDagger , and Stuart F. J. Le GriceDagger **

From the Dagger  Human Immunodeficiency Virus Drug Resistance Program, Division of Basic Sciences, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702,  Science Applications International Corporation, Frederick, Maryland 21702, and the || Department of Biological Chemistry, College of Medicine, University of California, Irvine, California 92697-1700

The 55-kDa reverse transcriptase (RT) domain of the Ty3 POL3 open reading frame was purified and evaluated on conformationally distinct nucleic acid duplexes. Purified enzyme migrated as a monomer by size exclusion chromatography. Enzymatic footprinting indicate Ty3 RT protects template nucleotides +7 through -21 and primer nucleotides -1 through -24. Contrary to previous data with retroviral enzymes, a 4-base pair region of the template-primer duplex remained nuclease accessible. The C-terminal portion of Ty3 RT encodes a functional RNase H domain, although the hydrolysis profile suggests an increased spatial separation between the catalytic centers. Despite conservation of catalytically important residues in the RNase H domain, Fe2+ fails to replace Mg2+ in the RNase H catalytic center for localized generation of hydroxyl radicals, again suggesting this domain may be structurally distinct from its retroviral counterparts. RNase H specificity was investigated using a model system challenging the enzyme to select the polypurine tract primer from within an RNA/DNA hybrid, extend this into (+) DNA, and excise the primer from nascent DNA. Purified RT catalyzed each of these three steps but was almost inactive on a non-polypurine tract RNA primer. Our studies provide the first detailed characterization of the enzymatic activities of a retrotransposon reverse transcriptase.

* This work was supported in part by National Institutes of Health Grants GM 52263 (to S. F. J. L. G.) and GM33281 (H. M. N.-M.).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.

§ These authors contributed equally to this work.

** To whom correspondence should be addressed: HIV Drug Resistance Program, Div. of Basic Sciences, NCI-FCRDC, Bldg. 535, Frederick, MD 21702. Tel.: 301-846-5256; Fax: 301-846-6013; E-mail: slegrice@mail.ncifcrf.gov.

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