Reference: Rausch JW, et al. (2000) Interaction of p55 reverse transcriptase from the Saccharomyces cerevisiae retrotransposon Ty3 with conformationally distinct nucleic acid duplexes. J Biol Chem 275(18):13879-87

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Abstract


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, Fe(2+) fails to replace Mg(2+) 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.

Reference Type
Journal Article
Authors
Rausch JW, Grice MK, Henrietta M, Nymark-McMahon, Miller JT, Le Grice SF
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