Interaction of p55 Reverse Transcriptase from theSaccharomyces cerevisiae Retrotransposon Ty3 with Conformationally Distinct Nucleic Acid Duplexes

• Published in: The Journal of biological chemistry 2000-05, Vol.275 (18), p.13879-13887
• Main Authors: Rausch, Jason W., Grice, Marion K.Bona-Le, Henrietta, M., Nymark, McMahon, Miller, Jennifer T., Le Grice, Stuart F.J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.18.13879

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.